Interferon-like protein zcyto21

ABSTRACT

The present invention relates to polynucleotide and polypeptide molecules for Zcyto21, an interferon-like protein, which is most closely related to interferon-α at the amino acid sequence level. The present invention also includes antibodies to the Zcyto21 polypeptides, and methods of using the polynucleotides and polypeptides.

The present application is a continuation of U.S. patent applicationSer. No. 11/548,023, filed Oct. 10, 2006, which is a divisional of U.S.patent application Ser. No. 10/927,815, filed Aug. 27, 2004, now U.S.Pat. No. 7,253,261, which is a divisional of U.S. patent applicationSer. No. 09/895,834, now U.S. Pat. No. 6,927,040, filed Jun. 29, 2001,which claims the benefit of U.S. Patent Application Ser. Nos.60/215,446, filed Jun. 30, 2000, and 60/285,424, filed Apr. 20, 2001,all of which are herein incorporated by reference.

BACKGROUND OF THE INVENTION

Cellular differentiation of multicellular organisms is controlled byhormones and polypeptide growth factors. These diffusable moleculesallow cells to communicate with each other and act in concert to formtissues and organs, and to repair and regenerate damaged tissue.Examples of hormones and growth factors include the steroid hormones,parathyroid hormone, follicle stimulating hormone, the interferons, theinterleukins, platelet derived growth factor, epidermal growth factor,and granulocyte-macrophage colony stimulating factor, among others.

Hormones and growth factors influence cellular metabolism by binding toreceptor proteins. Certain receptors are integral membrane proteins thatbind with the hormone or growth factor outside the cell, and that arelinked to signaling pathways within the cell, such as second messengersystems. Other classes of receptors are soluble intracellular molecules.

Cytokines generally stimulate proliferation or differentiation of cellsof the hematopoietic lineage or participate in the immune andinflammatory response mechanisms of the body. Examples of cytokineswhich affect hematopoiesis are erythropoietin (EPO), which stimulatesthe development of red blood cells; thrombopoietin (TPO), whichstimulates development of cells of the megakaryocyte lineage; andgranulocyte-colony stimulating factor (G-CSF), which stimulatesdevelopment of neutrophils. These cytokines are useful in restoringnormal blood cell levels in patients suffering from anemia,thrombocytopenia, and neutropenia or receiving chemotherapy for cancer.Cytokines play important roles in the regulation of hematopoiesis andimmune responses, and can influence lymphocyte development.

The human class II cytokine family includes interferon-α (IFN-α)subtypes, interferon-β (IFN-β), interferon-γ (IFN-γ), IL-10, IL-19 (U.S.Pat. No. 5,985,614), MDA-7 (Jiang et al., Oncogene 11, 2477-2486,(1995)), IL-20 (Jiang et al., Oncogene 11, 2477-2486, (1995)), IL-22(Xie et al., J. Biol. Chem. 275, 31335-31339, (2000)), and AK-155(Knappe et al., J. Virol. 74, 3881-3887, (2000)). Most cytokines bindand transduce signals through either Class I or Class II cytokinereceptors. Members of human class II cytokine receptor family includeinterferon-αR1 (IFN-αR1), interferon-γ-R2 (IFN-γ-R2), interferon-γ R1(IFN-γ R1), interferon-γR2 (IFN-γR2), IL-10R (Liu et al., J. Immunol.152, 1821-1829, (1994)), CRF2-4 (Lutfalla et al. Genomics 16, 366-373,(1993)), IL-20Rβ (Blumberg et al., Cell 104, 9-19, (2001)) (also knownas zcytor7 (U.S. Pat. No. 5,945,511) and CRF2-8 (Kotenko et al.,Oncogene 19, 2557-2565, (2000)), IL-20Rβ (Blumberg et al., ibid, (2001))(also known as DIRSI (PCT WO 99/46379)), IL-22RA1 (IL-22 receptor-α1,submitted to HUGO for approval) (also known as IL-22R (Xie et al., J.Biol. Chem. 275, 31335-31339, (2000)), zcytor11 (U.S. Pat. No.5,965,704) and CRF2-9 (Kotenko et al., Oncogene 19, 2557-2565, (2000)),and tissue factor.

Class II cytokine receptors are typically heterodimers composed of twodistinct receptor chains, the α and β receptor subunits (Stahl et al.,Cell 74, 587-590, (1993)). In general, the α subunits are the primarycytokine binding proteins, and the β subunits are required for formationof high affinity binding sites, as well as for signal transduction. Anexception is the IL-20 receptor in which both subunits are required forIL-20 binding (Blumberg et al., ibid, (2001)).

The class II cytokine receptors are identified by a conservedcytokine-binding domain of about 200 amino acids (D200) in theextracellular portion of the receptor. This cytokine-binding domain iscomprised of two fibronectin type III (FnIII) domains of approximately100 amino acids each (Bazan J. F. Proc. Natl. Acad. Sci. USA 87,6934-6938, (1990); Thoreau et al., FEBS Lett. 282, 16-31, (1991)). EachFnIII domain contains conserved Cys, Pro, and Trp residues thatdetermine a characteristic folding pattern of seven β-strands similar tothe constant domain of immunoglobulins (Uze et al., J. InterferonCytokine Res. 15, 3-26, (1995)). The conserved structural elements ofthe class II cytokine receptor family make it possible to identify newmembers of this family on the basis of primary amino acid sequencehomology. Previously we have successfully identified two new members ofclass II cytokine receptor family, zcytor7 (U.S. Pat. No. 5,945,511)(also known as IL-20R α (Blumberg et al., ibid, (2001)) and zcytor11(U.S. Pat. No. 5,965,704) (also known as IL-22R (Blumberg et al., ibid,(2001)), using this approach. Identification of additional novel membersof the class II cytokine receptor family is of interest becausecytokines play a vital role in regulating biological responses.

IL-22, also known as IL-TIF (IL-10-related T cell-derived induciblefactor) (Dumoutier et al., J. Immunology 164, 1814-1819, (2000)), is arecently described IL-10 homologue. Mouse IL-22 was originallyidentified as a gene induced by IL-9 in T cells and mast cells in vitro(Dumoutier et al., J. Immunology 164, 1814-1819, (2000)). Acute phasereactant induction activity was observed in mouse liver upon IL-22injection, and IL-22 expression was rapidly induced afterlipopolysaccharide (LPS) injection, suggesting that IL-22 contributes tothe inflammatory response in vivo (Dumoutier et al., Proc. Natl. Acad.Sci. U.S.A. 97, 10144-10149, (2000)).

The interleukins are a family of cytokines that mediate immunologicalresponses, including inflammation. The interleukins mediate a variety ofinflammatory pathologies. Central to an immune response is the T cell,which produce many cytokines and adaptive immunity to antigens.Cytokines produced by the T cell have been classified as type 1 and type2 (Kelso, A. Immun. Cell Biol. 76:300-317, 1998). Type 1 cytokinesinclude IL-2, IFN-γ, LT-α, and are involved in inflammatory responses,viral immunity, intracellular parasite immunity and allograft rejection.Type 2 cytokines include IL-4, IL-5, IL-6, IL-10 and IL-13, and areinvolved in humoral responses, helminth immunity and allergic response.Shared cytokines between Type 1 and 2 include IL-3, GM-CSF and TNF-α.There is some evidence to suggest that Type 1 and Type 2 producing Tcell populations preferentially migrate into different types of inflamedtissue.

Of particular interest, from a therapeutic standpoint, are theinterferons (reviews on interferons are provided by De Maeyer and DeMaeyer-Guignard, “Interferons,” in The Cytokine Handbook, 3^(rd)Edition, Thompson (ed.), pages 491-516 (Academic Press Ltd. 1998), andby Walsh, Biopharmaceuticals: Biochemistry and Biotechnology, pages158-188 (John Wiley & Sons 1998)). Interferons exhibit a variety ofbiological activities, and are useful for the treatment of certainautoimmune diseases, particular cancers, and the enhancement of theimmune response against infectious agents, including viruses, bacteria,fungi, and protozoa. To date, six forms of interferon have beenidentified, which have been classified into two major groups. Theso-called “type I” interferons include interferon-α, interferon-β,interferon-ω, interferon-δ, and interferon-τ. Currently, interferon-γand one subclass of interferon-α are the only type II interferons.

Type I interferons, which are thought to be derived from the sameancestral gene, have retained sufficient similar structure to act by thesame cell surface receptor. The α-chain of the human interferon-α/βreceptor comprises an extracellular N-terminal domain, which has thecharacteristics of a class II cytokine receptor. Interferon-γ does notshare significant homology with the type I interferons or with the typeII interferon-α subtype, but shares a number of biological activitieswith the type I interferons.

In humans, at least 16 non-allelic genes code for different subtypes ofinterferon-α, while interferons β and ω are encoded by single genes.Type I interferon genes are clustered in the short arm of chromosome 9.Unlike typical structural human genes, interferon-α, interferon-β, andinterferon-ω lack introns. A single gene for human interferon-γ islocalized on chromosome 12 and contains three introns. To date,interferon-τ has been described only in cattle and sheep, whileinterferon-δ has been described only in pigs.

Clinicians are taking advantage of the multiple activities ofinterferons by using the proteins to treat a wide range of conditions.For example, one form of interferon-α has been approved for use in morethan 50 countries for the treatment of medical conditions such as hairycell leukemia, renal cell carcinoma, basal cell carcinoma, malignantmelanoma, AIDS-related Kaposi's sarcoma, multiple myeloma, chronicmyelogenous leukemia, non-Hodgkin's lymphoma, laryngeal papillomatosis,mycosis fungoides, condyloma acuminata, chronic hepatitis B, hepatitisC, chronic hepatitis D, and chronic non-A, non-B/C hepatitis. The U.S.Food and Drug Administration has approved the use of interferon-β totreat multiple sclerosis, a chronic disease of the nervous system.Interferon-γ is used to treat chronic granulomatous diseases, in whichthe interferon enhances the patient's immune response to destroyinfectious bacterial, fungal, and protozoal pathogens. Clinical studiesalso indicate that interferon-γ may be useful in the treatment of AIDS,leishmaniasis, and lepromatous leprosy.

The demonstrated in vivo activities of the cytokine family illustratethe enormous clinical potential of, and need for, other cytokines,cytokine agonists, and cytokine antagonists. The present inventionaddresses these needs by providing a new cytokine that stimulates cellsof the hematopoietic cell lineage, as well as related compositions andmethods.

BRIEF DESCRIPTION OF THE FIGURES

The FIGURE is a Hopp/Woods hydrophilicity profile of the Zcyto21 proteinsequence shown in SEQ ID NO:2. The profile is based on a slidingsix-residue window. Buried G, S, and T residues and exposed H, Y, and Wresidues were ignored. These residues are indicated in the FIGURE bylower case letters.

DETAILED DESCRIPTION OF THE INVENTION

Prior to setting forth the invention in detail, it may be helpful to theunderstanding thereof to define the following terms:

The term “affinity tag” is used herein to denote a polypeptide segmentthat can be attached to a second polypeptide to provide for purificationor detection of the second polypeptide or provide sites for attachmentof the second polypeptide to a substrate. In principal, any peptide orprotein for which an antibody or other specific binding agent isavailable can be used as an affinity tag. Affinity tags include apoly-histidine tract, protein A (Nilsson et al., EMBO J. 4:1075, 1985;Nilsson et al., Methods Enzymol. 198:3, 1991), glutathione S transferase(Smith and Johnson, Gene 67:31, 1988), Glu-Glu affinity tag(Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952-4, 1985),substance P, Flag™ peptide (Hopp et al., Biotechnology 6:1204-10, 1988),streptavidin binding peptide, or other antigenic epitope or bindingdomain. See, in general, Ford et al., Protein Expression andPurification 2: 95-107, 1991. DNAs encoding affinity tags are availablefrom commercial suppliers (e.g., Pharmacia Biotech, Piscataway, N.J.).

The term “allelic variant” is used herein to denote any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inphenotypic polymorphism within populations. Gene mutations can be silent(no change in the encoded polypeptide) or may encode polypeptides havingaltered amino acid sequence. The term allelic variant is also usedherein to denote a protein encoded by an allelic variant of a gene.

The terms “amino-terminal” and “carboxyl-terminal” are used herein todenote positions within polypeptides. Where the context allows, theseterms are used with reference to a particular sequence or portion of apolypeptide to denote proximity or relative position. For example, acertain sequence positioned carboxyl-terminal to a reference sequencewithin a polypeptide is located proximal to the carboxyl terminus of thereference sequence, but is not necessarily at the carboxyl terminus ofthe complete polypeptide.

The term “complement/anti-complement pair” denotes non-identicalmoieties that form a non-covalently associated, stable pair underappropriate conditions. For instance, biotin and avidin (orstreptavidin) are prototypical members of a complement/anti-complementpair. Other exemplary complement/anti-complement pairs includereceptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs,sense/antisense polynucleotide pairs, and the like. Where subsequentdissociation of the complement/anti-complement pair is desirable, thecomplement/anti-complement pair preferably has a binding affinity of<10⁹ M⁻¹.

The term “complements of a polynucleotide molecule” denotes apolynucleotide molecule having a complementary base sequence and reverseorientation as compared to a reference sequence. For example, thesequence 5′ ATGCACGGG 3′ is complementary to 5′ CCCGTGCAT 3′.

The term “degenerate nucleotide sequence” denotes a sequence ofnucleotides that includes one or more degenerate codons (as compared toa reference polynucleotide molecule that encodes a polypeptide).Degenerate codons contain different triplets of nucleotides, but encodethe same amino acid residue (i.e., GAU and GAC triplets each encodeAsp).

The term “expression vector” is used to denote a DNA molecule, linear orcircular, that comprises a segment encoding a polypeptide of interestoperably linked to additional segments that provide for itstranscription. Such additional segments include promoter and terminatorsequences, and may also include one or more origins of replication, oneor more selectable markers, an enhancer, a polyadenylation signal, etc.Expression vectors are generally derived from plasmid or viral DNA, ormay contain elements of both.

The term “isolated”, when applied to a polynucleotide, denotes that thepolynucleotide has been removed from its natural genetic milieu and isthus free of other extraneous or unwanted coding sequences, and is in aform suitable for use within genetically engineered protein productionsystems. Such isolated molecules are those that are separated from theirnatural environment and include cDNA and genomic clones. Isolated DNAmolecules of the present invention are free of other genes with whichthey are ordinarily associated, but may include naturally occurring 5′and 3′ untranslated regions such as promoters and terminators. Theidentification of associated regions will be evident to one of ordinaryskill in the art (see for example, Dynan and Tijan, Nature 316:774-78,1985).

An “isolated” polypeptide or protein is a polypeptide or protein that isfound in a condition other than its native environment, such as apartfrom blood and animal tissue. In a preferred form, the isolatedpolypeptide is substantially free of other polypeptides, particularlyother polypeptides of animal origin. It is preferred to provide thepolypeptides in a highly purified form, i.e. greater than 95% pure, morepreferably greater than 99% pure. When used in this context, the term“isolated” does not exclude the presence of the same polypeptide inalternative physical forms, such as dimers or alternatively glycosylatedor derivatized forms.

The term “neoplastic”, when referring to cells, indicates cellsundergoing new and abnormal proliferation, particularly in a tissuewhere in the proliferation is uncontrolled and progressive, resulting ina neoplasm. The neoplastic cells can be either malignant, i.e. invasiveand metastatic, or benign.

The term “operably linked”, when referring to DNA segments, indicatesthat the segments are arranged so that they function in concert fortheir intended purposes, e.g., transcription initiates in the promoterand proceeds through the coding segment to the terminator.

The term “ortholog” denotes a polypeptide or protein obtained from onespecies that is the functional counterpart of a polypeptide or proteinfrom a different species. Sequence differences among orthologs are theresult of speciation.

“Paralogs” are distinct but structurally related proteins made by anorganism. Paralogs are believed to arise through gene duplication. Forexample, α-globin, β-globin, and myoglobin are paralogs of each other.

A “polynucleotide” is a single- or double-stranded polymer ofdeoxyribonucleotide or ribonucleotide bases read from the 5′ to the 3′end. Polynucleotides include RNA and DNA, and may be isolated fromnatural sources, synthesized in vitro, or prepared from a combination ofnatural and synthetic molecules. Sizes of polynucleotides are expressedas base pairs (abbreviated “bp”), nucleotides (“nt”), or kilobases(“kb”). Where the context allows, the latter two terms may describepolynucleotides that are single-stranded or double-stranded. When theterm is applied to double-stranded molecules it is used to denoteoverall length and will be understood to be equivalent to the term “basepairs”. It will be recognized by those skilled in the art that the twostrands of a double-stranded polynucleotide may differ slightly inlength and that the ends thereof may be staggered as a result ofenzymatic cleavage; thus all nucleotides within a double-strandedpolynucleotide molecule may not be paired.

A “polypeptide” is a polymer of amino acid residues joined by peptidebonds, whether produced naturally or synthetically. Polypeptides of lessthan about 10 amino acid residues are commonly referred to as“peptides”.

The term “promoter” is used herein for its art-recognized meaning todenote a portion of a gene containing DNA sequences that provide for thebinding of RNA polymerase and initiation of transcription. Promotersequences are commonly, but not always, found in the 5′ non-codingregions of genes.

A “protein” is a macromolecule comprising one or more polypeptidechains. A protein may also comprise non-peptidic components, such ascarbohydrate groups. Carbohydrates and other non-peptidic substituentsmay be added to a protein by the cell in which the protein is produced,and will vary with the type of cell. Proteins are defined herein interms of their amino acid backbone structures; substituents such ascarbohydrate groups are generally not specified, but may be presentnonetheless.

The term “receptor” denotes a cell-associated protein that binds to abioactive molecule (i.e., a ligand) and mediates the effect of theligand on the cell. Membrane-bound receptors are characterized by amulti-peptide structure comprising an extracellular ligand-bindingdomain and an intracellular effector domain that is typically involvedin signal transduction. Binding of ligand to receptor results in aconformational change in the receptor that causes an interaction betweenthe effector domain and other molecule(s) in the cell. This interactionin turn leads to an alteration in the metabolism of the cell. Metabolicevents that are linked to receptor-ligand interactions include genetranscription, phosphorylation, dephosphorylation, increases in cyclicAMP production, mobilization of cellular calcium, mobilization ofmembrane lipids, cell adhesion, hydrolysis of inositol lipids andhydrolysis of phospholipids. In general, receptors can be membranebound, cytosolic or nuclear; monomeric (e.g., thyroid stimulatinghormone receptor, beta-adrenergic receptor) or multimeric (e.g., PDGFreceptor, growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSFreceptor, erythropoietin receptor and IL-6 receptor).

The term “secretory signal sequence” denotes a DNA sequence that encodesa polypeptide (a “secretory peptide”) that, as a component of a largerpolypeptide, directs the larger polypeptide through a secretory pathwayof a cell in which it is synthesized. The larger polypeptide is commonlycleaved to remove the secretory peptide during transit through thesecretory pathway.

The term “splice variant” is used herein to denote alternative forms ofRNA transcribed from a gene. Splice variation arises naturally throughuse of alternative splicing sites within a transcribed RNA molecule, orless commonly between separately transcribed RNA molecules, and mayresult in several mRNAs transcribed from the same gene. Splice variantsmay encode polypeptides having altered amino acid sequence. The termsplice variant is also used herein to denote a protein encoded by asplice variant of an mRNA transcribed from a gene.

Molecular weights and lengths of polymers determined by impreciseanalytical methods (e.g., gel electrophoresis) will be understood to beapproximate values. When such a value is expressed as “about” X or“approximately” X, the stated value of X will be understood to beaccurate to 110%.

All references cited herein are incorporated by reference in theirentirety.

Zcyto21 gene encodes a polypeptide of 200 amino acids, as shown in SEQID NO:2. The signal sequence for Zcyto21 can be predicted as comprisingamino acid residue 1 (Met) through amino acid residue 19 (Ala) of SEQ IDNO:2. The mature peptide for Zcyto21 begins at amino acid residue 20(Gly).

The Zcyto21 gene is contained in BAC sequences AC011445, and AC018477which have been mapped to human chromosome 19q13.13. This region ofchromosome 19 may also comprise a cluster of interferon-like genes. Aconsensus cDNA showing a polynucleotide sequence of Zcyto21 is shown inSEQ ID NO:6 and the polypeptide it encodes is shown in SEQ ID NO:7.

As described below, the present invention provides isolated polypeptideshaving an amino acid sequence that is at least 70%, at least 80%, or atleast 90%, 95%, 96%, 97%, 98% or 99% identical to either amino acidresidues 20 to 200 of SEQ ID NO:2 or amino acid residues 1 to 200 of SEQID NO:2. The present invention also provides isolated polypeptideshaving an amino acid sequence that is at least 70%, at least 80%, or atleast 90%, 95%, 96%, 97%, 98% or 99% identical to either amino acidresidues 20 to 219 of SEQ ID NO:9 or amino acid residues 1 to 219 of SEQID NO:9. The present invention also provides isolated polypeptideshaving an amino acid sequence that is at least 70%, at least 80%, or atleast 90%, 95%, 96%, 97%, 98% or 99% identical to either amino acidresidues 20 to 203 of SEQ ID NO:12 or amino acid residues 1 to 203 ofSEQ ID NO:12. The present invention also includes a polypeptide thatfurther comprises a signal secretory sequence that resides in anamino-terminal position relative to the first amino acid sequence,wherein the signal secretory sequence comprises amino acid residues 1 to19 of the amino acid sequence of SEQ ID NO:2.

In general, cytokines are predicted to have a four-alpha helixstructure, with helices A, C and D being most important inligand-receptor interactions, and are more highly conserved amongmembers of the family. However, the interferons (INF), andinterferon-alpha and interferon-tau in particular, are characterized assix helix bundles. INF helix A is equivalent to helix A of Zcyto21; INFhelix B is equivalent to helix C of Zcyto21; INF helix C is equivalentto helix D of Zcyto21, and INF helix D is equivalent to helix F ofZcyto21. Thus, the loop between the AB loop, and CD loop of INF isexpanded in Zcyto21 to contain short helices B and E of Zcyto21.

Zcyto21 helices are predicted as follows: helix A is defined by aminoacid residues 49 (Ser) to 63 (Leu); helix B by amino acid residues 76(Asn) to 84 (Val); helix C by amino acid residues 89 (Val) to 104 (Ala);helix D by amino acid residues 111 (Glu) to 133 (Gln); helix E by aminoacid residues 137 (Thr) to 158 (Lys); and helix F by amino acid residues163 (Gly) to 189 (Leu); as shown in SEQ ID NO: 2. The cysteine residuesare conserved between Zcyto21, and INF-α, and may form an intermoleculardisulfide bond, in particular to form homodimers with additional Zcyto21molecules. Further analysis of Zcyto21 based on multiple alignmentspredicts that cysteines at amino acid residues 34 and 131, and 68 and164, (as shown in SEQ ID NO: 2) will form intramolecular disulfidebonds. The cysteine at residue 190 is free, and may form anintermolecular disulfide association. The corresponding polynucleotidesencoding the Zcyto21 polypeptide regions, domains, motifs, residues andsequences described herein are as shown in SEQ ID NO:1. The degeneratepolynucleotide sequence of SEQ ID NO:2 is shown in SEQ ID NO:3. Thedegenerate polynucleotide sequence of SEQ ID NO:9 is shown in SEQ IDNO:10. The degenerate polynucleotide sequence of SEQ ID NO:12 is shownin SEQ ID NO:13.

Detailed mutational analysis of murine IL-2 (Zurawski et al., EMBO J.12:5113-5119, 1993) shows residues in helices A and C are important forbinding to IL-2Rβ; critical residues are Asp₃₄, Asn₉₉, and Asn₁₀₃.Multiple residues within murine IL-2 loop A/B and helix B are importantfor IL-2Rα binding, while only a single residue, Gln₁₄₁ in helix D, isvital for binding with IL-2Rα. Similarly, helices A and C are sites ofinteraction between IL-4 and IL-4Rα (the structurally similar toIL-2Rα), and residues within helix D are vital for IL-2Rα interaction(Wang et al., Proc. Natl. Acad. Sci. USA 94:1657-1662, 1997; Kruse etal., EMBO J. 11:3237-3244, 1992). In particular, the mutation Tyr₁₂₄ toAsp in human IL-4 creates an antagonist, which binds with IL-4Rα but notIL-2Rα and therefore cannot signal (Kruse et al. ibid. 1992).

Four-helical bundle cytokines are also grouped by the length of theircomponent helices. “Long-helix” form cytokines generally consist ofbetween 24-30 residue helices, and include IL-6, ciliary neutrotrophicfactor (CNTF), leukemia inhibitory factor (LIF) and human growth hormone(hGH). “Short-helix” form cytokines generally consist of between 18-21residue helices and include IL-2, IL-4 and GM-CSF. Studies using CNTFand IL-6 demonstrated that a CNTF helix can be exchanged for theequivalent helix in IL-6, conferring CTNF-binding properties to thechimera. Thus, it appears that functional domains of four-helicalcytokines are determined on the basis of structural homology,irrespective of sequence identity, and can maintain functional integrityin a chimera (Kallen et al., J. Biol. Chem. 274:11859-11867, 1999).Therefore, the helical domains of Zcyto21 will be useful for preparingchimeric fusion molecules, particularly with other interferons todetermine and modulate receptor binding specificity. Of particularinterest are fusion proteins that combine helical and loop domains frominterferons and cytokines such as INF-α, IL-10, human growth hormone.

Zcyto21 mRNA has been identified in tissues of brain, islet, prostate,testis, pituitary, placenta, ovarian tumor lung tumor, rectal tumor andovarian tumor, as well as an activated immune cell line (CD3+) and aprostate epithelial cell line, which had been transformed with humanpapilloma virus IV (HPVS).

The present invention provides polynucleotide molecules, including DNAand RNA molecules, that encode the Zcyto21 polypeptides disclosedherein. Those skilled in the art will readily recognize that, in view ofthe degeneracy of the genetic code, considerable sequence variation ispossible among these polynucleotide molecules. SEQ ID NOs:3, 10, and 13are degenerate DNA sequences that encompasses all DNAs that encode theZcyto21polypeptide of SEQ ID NOs:2, 9, and 12, respectively. Thoseskilled in the art will recognize that the degenerate sequence of SEQ IDNO:3, for example, also provides all RNA sequences encoding SEQ ID NO:2by substituting U for T. Thus, Zcyto21 polypeptide-encodingpolynucleotides comprising nucleotide 1 or 58 to nucleotide 603 of SEQID NO:3 and their RNA equivalents are contemplated by the presentinvention. Table 1 sets forth the one-letter codes used within SEQ IDNO:3 to denote degenerate nucleotide positions. “Resolutions” are thenucleotides denoted by a code letter. “Complement” indicates the codefor the complementary nucleotide(s). For example, the code Y denoteseither C or T, and its complement R denotes A or G, with A beingcomplementary to T, and G being complementary to C. TABLE 1 NucleotideResolution Complement Resolution A A T T C C G G G G C C T T A A R A|G YC|T Y C|T R A|G M A|C K G|T K G|T M A|C S C|G S C|G W A|T W A|T H A|C|TD A|G|T B C|G|T V A|C|G V A|C|G B C|G|T D A|G|T H A|C|T N A|C|G|T NA|C|G|T

The degenerate codons used in SEQ ID NOs:3, 10, and 13, encompassing allpossible codons for a given amino acid, are set forth in Table 2. TABLE2 One Amino Letter Degenerate Acid Code Codons Codon Cys C TGC TGT TGYSer S AGC AGT TCA TCC TCG TCT WSN Thr T ACA ACC ACG ACT ACN Pro P CCACCC CCG CCT CCN Ala A GCA GCC GCG GCT GCN Gly G GGA GGC GGG GGT GGN AsnN AAC AAT AAY Asp D GAC GAT GAY Glu E GAA GAG GAR Gln Q CAA CAG CAR HisH CAC CAT CAY Arg R AGA AGG CGA CGC CGG CGT MGN Lys K AAA AAG AAR Met MATG ATG Ile I ATA ATC ATT ATH Leu L CTA CTC CTG CTT TTA TTG YTN Val VGTA GTC GTG GTT GTN Phe F TTC TTT TTY Tyr Y TAC TAT TAY Trp W TGG TGGTer · TAA TAG TGA TRR Asn|Asp B RAY Glu|Gln Z SAR Any X NNN

One of ordinary skill in the art will appreciate that some ambiguity isintroduced in determining a degenerate codon, representative of allpossible codons encoding each amino acid. For example, the degeneratecodon for serine (WSN) can, in some circumstances, encode arginine(AGR), and the degenerate codon for arginine (MGN) can, in somecircumstances, encode serine (AGY). A similar relationship existsbetween codons encoding phenylalanine and leucine. Thus, somepolynucleotides encompassed by the degenerate sequence may encodevariant amino acid sequences, but one of ordinary skill in the art caneasily identify such variant sequences by reference to the amino acidsequence of SEQ ID NO:2. Variant sequences can be readily tested forfunctionality as described herein.

One of ordinary skill in the art will also appreciate that differentspecies can exhibit “preferential codon usage.” In general, see,Grantham, et al., Nuc. Acids Res. 8:1893-912, 1980; Haas, et al. Curr.Biol. 6:315-24, 1996; Wain-Hobson, et al., Gene 13:355-64, 1981;Grosjean and Fiers, Gene 18:199-209, 1982; Holm, Nuc. Acids Res.14:3075-87, 1986; Ikemura, J. Mol. Biol. 158:573-97, 1982. As usedherein, the term “preferential codon usage” or “preferential codons” isa term of art referring to protein translation codons that are mostfrequently used in cells of a certain species, thus favoring one or afew representatives of the possible codons encoding each amino acid (SeeTable 3). For example, the amino acid Threonine (Thr) may be encoded byACA, ACC, ACG, or ACT, but in mammalian cells ACC is the most commonlyused codon; in other species, for example, insect cells, yeast, virusesor bacteria, different Thr codons may be preferential. Preferentialcodons for a particular species can be introduced into thepolynucleotides of the present invention by a variety of methods knownin the art. Introduction of preferential codon sequences intorecombinant DNA can, for example, enhance production of the protein bymaking protein translation more efficient within a particular cell typeor species. Therefore, the degenerate codon sequence disclosed in SEQ IDNO:3 serves as a template for optimizing expression of polynucleotidesin various cell types and species commonly used in the art and disclosedherein. Sequences containing preferential codons can be tested andoptimized for expression in various species, and tested forfunctionality as disclosed herein.

As previously noted, the isolated polynucleotides of the presentinvention include DNA and RNA. Methods for preparing DNA and RNA arewell known in the art. In general, RNA is isolated from a tissue or cellthat produces large amounts of Zcyto21RNA. Such tissues and cells areidentified by Northern blotting (Thomas, Proc. Natl. Acad. Sci. USA77:5201, 1980), or by screening conditioned medium from various celltypes for activity on target cells or tissue. Once the activity or RNAproducing cell or tissue is identified, total RNA can be prepared usingguanidinium isothiocyanate extraction followed by isolation bycentrifugation in a CsCl gradient (Chirgwin et al., Biochemistry18:52-94, 1979). Poly (A)⁺ RNA is prepared from total RNA using themethod of Aviv and Leder (Proc. Natl. Acad. Sci. USA 69:1408-12, 1972).Complementary DNA (cDNA) is prepared from poly(A)⁺ RNA using knownmethods. In the alternative, genomic DNA can be isolated.Polynucleotides encoding Zcyto21 polypeptides are then identified andisolated by, for example, hybridization or PCR.

A longer clone encoding Zcyto21 can be obtained by conventional cloningprocedures. Complementary DNA (cDNA) clones are preferred, although forsome applications (e.g., expression in transgenic animals) it may bepreferable to use a genomic clone, or to modify a cDNA clone to includeat least one genomic intron. Methods for preparing cDNA and genomicclones are well known and within the level of ordinary skill in the art,and include the use of the sequence disclosed herein, or parts thereof,for probing or priming a library. Expression libraries can be probedwith antibodies to Zcyto21 receptor fragments, or other specific bindingpartners.

The present invention further provides counterpart polypeptides andpolynucleotides from other species (orthologs). These species include,but are not limited to mammalian, avian, amphibian, reptile, fish,insect and other vertebrate and invertebrate species. Of particularinterest are Zcyto21 polypeptides from other mammalian species,including murine, porcine, ovine, bovine, canine, feline, equine, andother primate polypeptides. Orthologs of human Zcyto21 can be clonedusing information and compositions provided by the present invention incombination with conventional cloning techniques. For example, a cDNAcan be cloned using mRNA obtained from a tissue or cell type thatexpresses Zcyto21 as disclosed herein. Suitable sources of mRNA can beidentified by probing Northern blots with probes designed from thesequences disclosed herein. A library is then prepared from mRNA of apositive tissue or cell line. A Zcyto21-encoding cDNA can then beisolated by a variety of methods, such as by probing with a complete orpartial human cDNA or with one or more sets of degenerate probes basedon the disclosed sequences. A cDNA can also be cloned using thepolymerase chain reaction, or PCR (Mullis, U.S. Pat. No. 4,683,202),using primers designed from the representative human Zcyto21sequencedisclosed herein. Within an additional method, the cDNA library can beused to transform or transfect host cells, and expression of the cDNA ofinterest can be detected with an antibody to Zcyto21 polypeptide,binding studies or activity assays. Similar techniques can also beapplied to the isolation of genomic clones.

Those skilled in the art will recognize that the sequence disclosed inSEQ ID NO:1 represents a single allele of human Zcyto21 band thatallelic variation and alternative splicing are expected to occur.Allelic variants of this sequence can be cloned by probing cDNA orgenomic libraries from different individuals according to standardprocedures. Allelic variants of the DNA sequence shown in SEQ ID NO:1,including those containing silent mutations and those in which mutationsresult in amino acid sequence changes, are within the scope of thepresent invention, as are proteins which are allelic variants of SEQ IDNO:2. cDNAs generated from alternatively spliced mRNAs, which retain theproperties of the Zcyto21 polypeptide, are included within the scope ofthe present invention, as are polypeptides encoded by such cDNAs andmRNAs. Allelic variants and splice variants of these sequences can becloned by probing cDNA or genomic libraries from different individualsor tissues according to standard procedures known in the art. Examplesof alternatively spliced variants are shown in SEQ ID NO:8 (SEQ ID NO:9for the corresponding polypeptide), and in SEQ ID NO:11 (SEQ ID NO:12for the corresponding polypeptide). An example of an allelic variant isshown in SEQ ID NO:4, which corresponds to the polypeptide sequence asshown in SEQ ID NO:5. There is a polymorphism between the polypeptidesequence as shown in SEQ ID NO:1 and that shown in SEQ ID NO:4 atnucleotide number 572. This polymorphism might create an antagonist ofZcyto21 or a molecule of reduced or altered function, which might leadto a higher likelihood of disease susceptibility.

The present invention also provides reagents, which will find use indiagnostic applications. For example, the Zcyto21 gene, a probecomprising Zcyto21 DNA or RNA or a subsequence thereof can be used todetermine if the Zcyto21 gene is present on a human chromosome, such aschromosome 19, or if a gene mutation has occurred. Zcyto21 is located atthe q13.13 region of chromosome 19. Detectable chromosomal aberrationsat the Zcyto21 gene locus include, but are not limited to, aneuploidy,gene copy number changes, loss of heterogeneity (LOH), translocations,insertions, deletions, restriction site changes and rearrangements. Suchaberrations can be detected using polynucleotides of the presentinvention by employing molecular genetic techniques, such as restrictionfragment length polymorphism (RFLP) analysis, short tandem repeat (STR)analysis employing PCR techniques, and other genetic linkage analysistechniques known in the art (Sambrook et al., ibid.; Ausubel et. al.,ibid.; Marian, Chest 108:255-65, 1995).

The precise knowledge of a gene's position can be useful for a number ofpurposes, including: 1) determining if a sequence is part of an existingcontig and obtaining additional surrounding genetic sequences in variousforms, such as YACs, BACs or cDNA clones; 2) providing a possiblecandidate gene for an inheritable disease which shows linkage to thesame chromosomal region; and 3) cross-referencing model organisms, suchas mouse, which may aid in determining what function a particular genemight have.

For example, Delague et al., (Am. J. Hum. Genet. 67: 236-243, 2000)identified that Charcot-Marie-Tooth disease is localized to 19q13.1-13.3(Delague et al., Am. J. Hum. Genet. 67: 236-243, 2000).

A diagnostic could assist physicians in determining the type of diseaseand appropriate associated therapy, or assistance in genetic counseling.As such, the inventive anti-Zcyto21 antibodies, polynucleotides, andpolypeptides can be used for the detection of Zcyto21 polypeptide, mRNAor anti-Zcyto21 antibodies, thus serving as markers and can be directlyused for detecting or genetic diseases or cancers, as described herein,using methods known in the art and described herein. Further, Zcyto21polynucleotide probes can be used to detect abnormalities or genotypesassociated with chromosome 19 deletions and translocations associatedwith human diseases or other translocations involved with malignantprogression of tumors or other 19q13.13 mutations, which are expected tobe involved in chromosome rearrangements in malignancy; or in othercancers. Similarly, Zcyto21 polynucleotide probes can be used to detectabnormalities or genotypes associated with chromosome 19q13.13 trisomyand chromosome loss associated with human diseases or spontaneousabortion. Thus, Zcyto21 polynucleotide probes can be used to detectabnormalities or genotypes associated with these defects.

In general, the diagnostic methods used in genetic linkage analysis, todetect a genetic abnormality or aberration in a patient, are known inthe art. Analytical probes will be generally at least 20 nt in length,although somewhat shorter probes can be used (e.g., 14-17 nt). PCRprimers are at least 5 nt in length, preferably 15 or more, morepreferably 20-30 nt. For gross analysis of genes, or chromosomal DNA, aZcyto21 polynucleotide probe may comprise an entire exon or more. Exonsare readily determined by one of skill in the art by comparing Zcyto21sequences (SEQ ID NO:1) with the genomic DNA for Zcyto21. In general,the diagnostic methods used in genetic linkage analysis, to detect agenetic abnormality or aberration in a patient, are known in the art.Most diagnostic methods comprise the steps of (a) obtaining a geneticsample from a potentially diseased patient, diseased patient orpotential non-diseased carrier of a recessive disease allele; (b)producing a first reaction product by incubating the genetic sample witha Zcyto21 polynucleotide probe wherein the polynucleotide will hybridizeto complementary polynucleotide sequence, such as in RFLP analysis or byincubating the genetic sample with sense and antisense primers in a PCRreaction under appropriate PCR reaction conditions; (iii) Visualizingthe first reaction product by gel electrophoresis and/or other knownmethod such as visualizing the first reaction product with a Zcyto21polynucleotide probe wherein the polynucleotide will hybridize to thecomplementary polynucleotide sequence of the first reaction; and (iv)comparing the visualized first reaction product to a second controlreaction product of a genetic sample from wild type patient. Adifference between the first reaction product and the control reactionproduct is indicative of a genetic abnormality in the diseased orpotentially diseased patient, or the presence of a heterozygousrecessive carrier phenotype for a non-diseased patient, or the presenceof a genetic defect in a tumor from a diseased patient, or the presenceof a genetic abnormality in a fetus or pre-implantation embryo. Forexample, a difference in restriction fragment pattern, length of PCRproducts, length of repetitive sequences at the Zcyto21 genetic locus,and the like, are indicative of a genetic abnormality, geneticaberration, or allelic difference in comparison to the normal wild typecontrol. Controls can be from unaffected family members, or unrelatedindividuals, depending on the test and availability of samples. Geneticsamples for use within the present invention include genomic DNA, mRNA,and cDNA isolated form any tissue or other biological sample from apatient, such as but not limited to, blood, saliva, semen, embryoniccells, amniotic fluid, and the like. The polynucleotide probe or primercan be RNA or DNA, and will comprise a portion of SEQ ID NO:1, thecomplement of SEQ ID NO:1, or an RNA equivalent thereof. Such methods ofshowing genetic linkage analysis to human disease phenotypes are wellknown in the art. For reference to PCR based methods in diagnostics seesee, generally, Mathew (ed.), Protocols in Human Molecular Genetics(Humana Press, Inc. 1991), White (ed.), PCR Protocols: Current Methodsand Applications (Humana Press, Inc. 1993), Cotter (ed.), MolecularDiagnosis of Cancer (Humana Press, Inc. 1996), Hanausek and Walaszek(eds.), Tumor Marker Protocols (Humana Press, Inc. 1998), Lo (ed.),Clinical Applications of PCR (Humana Press, Inc. 1998), and Meltzer(ed.), PCR in Bioanalysis (Humana Press, Inc. 1998)).

Mutations associated with the Zcyto21 locus can be detected usingnucleic acid molecules of the present invention by employing standardmethods for direct mutation analysis, such as restriction fragmentlength polymorphism analysis, short tandem repeat analysis employing PCRtechniques, amplification-refractory mutation system analysis,single-strand conformation polymorphism detection, RNase cleavagemethods, denaturing gradient gel electrophoresis, fluorescence-assistedmismatch analysis, and other genetic analysis techniques known in theart (see, for example, Mathew (ed.), Protocols in Human MolecularGenetics (Humana Press, Inc. 1991), Marian, Chest 108:255 (1995),Coleman and Tsongalis, Molecular Diagnostics (Human Press, Inc. 1996),Elles (ed.) Molecular Diagnosis of Genetic Diseases (Humana Press, Inc.1996), Landegren (ed.), Laboratory Protocols for Mutation Detection(Oxford University Press 1996), Birren et al (eds.), Genome Analysis,Vol 2: Detecting Genes (Cold Spring Harbor Laboratory Press 1998),Dracopoli et al. (eds.), Current Protocols in Human Genetics (John Wiley& Sons 1998), and Richards and Ward, “Molecular Diagnostic Testing,” inPrinciples of Molecular Medicine, pages 83-88 (Humana Press, Inc.1998)). Direct analysis of an Zcyto21 gene for a mutation can beperformed using a subject's genomic DNA. Methods for amplifying genomicDNA, obtained for example from peripheral blood lymphocytes, arewell-known to those of skill in the art (see, for example, Dracopoli etal (eds.), Current Protocols in Human Genetics, at pages 7.1.6 to 7.1.7(John Wiley & Sons 1998)).

Within embodiments of the invention, isolated Zcyto21-encoding nucleicacid molecules can hybridize under stringent conditions to nucleic acidmolecules having the nucleotide sequence of SEQ ID NO:1, to nucleic acidmolecules having the nucleotide sequence of nucleotides 58 to 603 of SEQID NO:1, or to nucleic acid molecules having a nucleotide sequencecomplementary to SEQ ID NO:1. In general, stringent conditions areselected to be about 5° C. lower than the thermal melting point (T_(m))for the specific sequence at a defined ionic strength and pH. The T_(m)is the temperature (under defined ionic strength and pH) at which 50% ofthe target sequence hybridizes to a perfectly matched probe.

A pair of nucleic acid molecules, such as DNA-DNA, RNA-RNA and DNA-RNA,can hybridize if the nucleotide sequences have some degree ofcomplementarity. Hybrids can tolerate mismatched base pairs in thedouble helix, but the stability of the hybrid is influenced by thedegree of mismatch. The T_(m) of the mismatched hybrid decreases by 1°C. for every 1-1.5% base pair mismatch. Varying the stringency of thehybridization conditions allows control over the degree of mismatch thatwill be present in the hybrid. The degree of stringency increases as thehybridization temperature increases and the ionic strength of thehybridization buffer decreases.

It is well within the abilities of one skilled in the art to adapt theseconditions for use with a particular polynucleotide hybrid. The T_(m)for a specific target sequence is the temperature (under definedconditions) at which 50% of the target sequence will hybridize to aperfectly matched probe sequence. Those conditions which influence theT_(m) include, the size and base pair content of the polynucleotideprobe, the ionic strength of the hybridization solution, and thepresence of destabilizing agents in the hybridization solution. Numerousequations for calculating T_(m) are known in the art, and are specificfor DNA, RNA and DNA-RNA hybrids and polynucleotide probe sequences ofvarying length (see, for example, Sambrook et al, Molecular Cloning: ALaboratory Manual, Second Edition (Cold Spring Harbor Press 1989);Ausubel et al., (eds.), Current Protocols in Molecular Biology (JohnWiley and Sons, Inc. 1987); Berger and Kimmel (eds.), Guide to MolecularCloning Techniques, (Academic Press, Inc. 1987); and Wetmur, Crit. Rev.Biochem. Mol. Biol. 26:227 (1990)). Sequence analysis software such asOLIGO 6.0 (LSR; Long Lake, Minn.) and Primer Premier 4.0 (PremierBiosoft International; Palo Alto, Calif.), as well as sites on theInternet, are available tools for analyzing a given sequence andcalculating T_(m) based on user defined criteria. Such programs can alsoanalyze a given sequence under defined conditions and identify suitableprobe sequences. Typically, hybridization of longer polynucleotidesequences, >50 base pairs, is performed at temperatures of about 20-25°C. below the calculated T_(m). For smaller probes, <50 base pairs,hybridization is typically carried out at the T_(m) or 5-10° C. belowthe calculated T_(m). This allows for the maximum rate of hybridizationfor DNA-DNA and DNA-RNA hybrids.

Following hybridization, the nucleic acid molecules can be washed toremove non-hybridized nucleic acid molecules under stringent conditions,or under highly stringent conditions. Typical stringent washingconditions include washing in a solution of 0.5×-2×SSC with 0.1% sodiumdodecyl sulfate (SDS) at 55-65° C. That is, nucleic acid moleculesencoding a variant Zcyto21 polypeptide hybridize with a nucleic acidmolecule having the nucleotide sequence of SEQ ID NO:1 (or itscomplement) under stringent washing conditions, in which the washstringency is equivalent to 0.5×-2×SSC with 0.1% SDS at 55-65° C.,including 0.5×SSC with 0.1% SDS at 55° C., or 2×SSC with 0.1% SDS at 65°C. One of skill in the art can readily devise equivalent conditions, forexample, by substituting SSPE for SSC in the wash solution.

Typical highly stringent washing conditions include washing in asolution of 0.1×-0.2×SSC with 0.1% sodium dodecyl sulfate (SDS) at50-65° C. In other words, nucleic acid molecules encoding a variantZcyto21 polypeptide hybridize with a nucleic acid molecule having thenucleotide sequence of SEQ ID NO:1 (or its complement) under highlystringent washing conditions, in which the wash stringency is equivalentto 0.1×-0.2×SSC with 0.1% SDS at 50-65° C., including 0.1×SSC with 0.1%SDS at 50° C., or 0.2×SSC with 0.1% SDS at 65° C.

The present invention also provides isolated Zcyto21 polypeptides thathave a substantially similar sequence identity to the polypeptides ofSEQ ID NO:2, or their orthologs. The term “substantially similarsequence identity” is used herein to denote polypeptides comprising atleast 70%, at least 80%, at least 90%, at least 95%, or greater than95%, 96%, 97%, 98%, or 99% sequence identity to the sequences shown inSEQ ID NO:2, or their orthologs. The present invention also includespolypeptides that comprise an amino acid sequence having at least 70%,at least 80%, at least 90%, at least 95% or greater than 95%, 96%, 97%,98%, or 99% sequence identity to the sequence of amino acid residues 1to 200 or 20 to 200 of SEQ ID NO:2. The present invention furtherincludes nucleic acid molecules that encode such polypeptides. Methodsfor determining percent identity are described below.

The present invention also contemplates variant Zcyto21 nucleic acidmolecules that can be identified using two criteria: a determination ofthe similarity between the encoded polypeptide with the amino acidsequence of SEQ ID NO:2, and/or a hybridization assay, as describedabove. Such Zcyto21 variants include nucleic acid molecules: (1) thathybridize with a nucleic acid molecule having the nucleotide sequence ofSEQ ID NO:1 (or its complement) under stringent washing conditions, inwhich the wash stringency is equivalent to 0.5×-2×SSC with 0.1% SDS at55-65° C.; or (2) that encode a polypeptide having at least 70%, atleast 80%, at least 90%, at least 95% or greater than 95%, 96%, 97%,98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO:2.Alternatively, Zcyto21 variants can be characterized as nucleic acidmolecules: (1) that hybridize with a nucleic acid molecule having thenucleotide sequence of SEQ ID NO:1 (or its complement) under highlystringent washing conditions, in which the wash stringency is equivalentto 0.1×-0.2×SSC with 0.1% SDS at 50-65° C.; and (2) that encode apolypeptide having at least 70%, at least 80%, at least 90%, at least95% or greater than 95% sequence identity to the amino acid sequence ofSEQ ID NO:2.

Percent sequence identity is determined by conventional methods. See,for example, Altschul et al., Bull. Math. Bio. 48:603 (1986), andHenikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1992).Briefly, two amino acid sequences are aligned to optimize the alignmentscores using a gap opening penalty of 10, a gap extension penalty of 1,and the “BLOSUM62” scoring matrix of Henikoff and Henikoff (ibid.) asshown in Table 3 (amino acids are indicated by the standard one-lettercodes).$\frac{{Total}\quad{number}\quad{of}\quad{identical}\quad{matches}}{\begin{matrix}\left\lbrack {{length}\quad{of}\quad{the}\quad{longer}\quad{sequence}\quad{plus}\quad{the}} \right. \\{{number}\quad{of}\quad{gaps}\quad{introduced}\quad{into}\quad{the}\quad{longer}} \\\left. \quad{{sequence}\quad{in}\quad{order}\quad{to}\quad{align}\quad{the}\quad{two}\quad{sequences}} \right\rbrack\end{matrix}} \times 100$ TABLE 3 A R N D C Q E G H I L K M F P S T W YV R −1 5 N −2 0 6 D −2 −2 1 6 C 0 −3 −3 −3 9 Q −1 1 0 0 −3 5 E −1 0 0 2−4 2 5 G 0 −2 0 −1 −3 −2 −2 6 H −2 0 1 −1 −3 0 0 −2 8 I −1 −3 −3 −3 −1−3 −3 −4 −3 4 L −1 −2 −3 −4 −1 −2 −3 −4 −3 2 4 K −1 2 0 −1 −3 1 1 −2 −1−3 −2 5 M −1 −1 −2 −3 −1 0 −2 −3 −2 1 2 −1 5 F −2 −3 −3 −3 −2 −3 −3 −3−1 0 0 −3 0 6 P −1 −2 −2 −1 −3 −1 −1 −2 −2 −3 −3 −1 −2 −4 7 S 1 −1 1 0−1 0 0 0 −1 −2 −2 0 −1 −2 −1 4 T 0 −1 0 −1 −1 −1 −1 −2 −2 −1 −1 −1 −1 −2−1 1 5 W −3 −3 −4 −4 −2 −2 −3 −2 −2 −3 −2 −3 −1 1 −4 −3 −2 11 Y −2 −2 −2−3 −2 −1 −2 −3 2 −1 −1 −2 −1 3 −3 −2 −2 2 7 V 0 −3 −3 −3 −1 −2 −2 −3 −33 1 −2 1 −1 −2 −2 0 −3 −1 4

Those skilled in the art appreciate that there are many establishedalgorithms available to align two amino acid sequences. The “FASTA”similarity search algorithm of Pearson and Lipman is a suitable proteinalignment method for examining the level of identity shared by an aminoacid sequence disclosed herein and the amino acid sequence of a putativevariant Zcyto21. The FASTA algorithm is described by Pearson and Lipman,Proc. Nat'l Acad. Sci. USA 85:2444 (1988), and by Pearson, Meth.Enzymol. 183:63 (1990).

Briefly, FASTA first characterizes sequence similarity by identifyingregions shared by the query sequence (e.g., SEQ ID NO:2) and a testsequence that have either the highest density of identities (if the ktupvariable is 1) or pairs of identities (if ktup=2), without consideringconservative amino acid substitutions, insertions, or deletions. The tenregions with the highest density of identities are then rescored bycomparing the similarity of all paired amino acids using an amino acidsubstitution matrix, and the ends of the regions are “trimmed” toinclude only those residues that contribute to the highest score. Ifthere are several regions with scores greater than the “cutoff” value(calculated by a predetermined formula based upon the length of thesequence and the ktup value), then the trimmed initial regions areexamined to determine whether the regions can be joined to form anapproximate alignment with gaps. Finally, the highest scoring regions ofthe two amino acid sequences are aligned using a modification of theNeedleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol. Biol.48:444 (1970); Sellers, SIAM J. Appl. Math. 26:787 (1974)), which allowsfor amino acid insertions and deletions. Preferred parameters for FASTAanalysis are: ktup=1, gap opening penalty=10, gap extension penalty=1,and substitution matrix=BLOSUM62. These parameters can be introducedinto a FASTA program by modifying the scoring matrix file (“SMATRIX”),as explained in Appendix 2 of Pearson, Meth. Enzymol. 183:63 (1990).

FASTA can also be used to determine the sequence identity of nucleicacid molecules using a ratio as disclosed above. For nucleotide sequencecomparisons, the ktup value can range between one to six, preferablyfrom three to six, most preferably three, with other parameters set asdefault.

Variant Zcyto21 polypeptides or polypeptides with substantially similarsequence identity are characterized as having one or more amino acidsubstitutions, deletions or additions. These changes are preferably of aminor nature, that is conservative amino acid substitutions (see Table4) and other substitutions that do not significantly affect the foldingor activity of the polypeptide; small deletions, typically of one toabout 30 amino acids; and amino- or carboxyl-terminal extensions, suchas an amino-terminal methionine residue, a small linker peptide of up toabout 20-25 residues, or an affinity tag. The present invention thusincludes polypeptides of from about 149 to 230 amino acid residues thatcomprise a sequence that is at least 70%, preferably at least 90%, andmore preferably 95%, 96%, 97%, 98%, 99% or more identical to thecorresponding region of SEQ ID NO:2. Polypeptides comprising affinitytags can further comprise a proteolytic cleavage site between theZcyto21 polypeptide and the affinity tag. Preferred such sites includethrombin cleavage sites and factor Xa cleavage sites. TABLE 4Conservative amino acid substitutions Basic: arginine lysine histidineAcidic: glutamic acid aspartic acid Polar: glutamine asparagineHydrophobic: leucine isoleucine valine Aromatic: phenylalaninetryptophan tyrosine Small: glycine alanine serine threonine methionine

Determination of amino acid residues that comprise regions or domainsthat are critical to maintaining structural integrity can be determined.Within these regions one can determine specific residues that will bemore or less tolerant of change and maintain the overall tertiarystructure of the molecule. Methods for analyzing sequence structureinclude, but are not limited to alignment of multiple sequences withhigh amino acid or nucleotide identity, secondary structurepropensities, binary patterns, complementary packing and buried polarinteractions (Barton, Current Opin. Struct. Biol. 5:372-376, 1995 andCordes et al., Current Opin. Struct. Biol. 6:3-10, 1996). In general,when designing modifications to molecules or identifying specificfragments determination of structure will be accompanied by evaluatingactivity of modified molecules.

Amino acid sequence changes are made in Zcyto21 polypeptides so as tominimize disruption of higher order structure essential to biologicalactivity. For example, where the Zcyto21 polypeptide comprises one ormore helices, changes in amino acid residues will be made so as not todisrupt the helix geometry and other components of the molecule wherechanges in conformation abate some critical function, for example,binding of the molecule to its binding partners. The effects of aminoacid sequence changes can be predicted by, for example, computermodeling as disclosed above or determined by analysis of crystalstructure (see, e.g., Lapthorn et al., Nat. Struct. Biol. 2:266-268,1995). Other techniques that are well known in the art compare foldingof a variant protein to a standard molecule (e.g., the native protein).For example, comparison of the cysteine pattern in a variant andstandard molecules can be made. Mass spectrometry and chemicalmodification using reduction and alkylation provide methods fordetermining cysteine residues which are associated with disulfide bondsor are free of such associations (Bean et al., Anal. Biochem.201:216-226, 1992; Gray, Protein Sci. 2:1732-1748, 1993; and Pattersonet al., Anal. Chem. 66:3727-3732, 1994). It is generally believed thatif a modified molecule does not have the same cysteine pattern as thestandard molecule folding would be affected. Another well known andaccepted method for measuring folding is circular dichrosism (CD).Measuring and comparing the CD spectra generated by a modified moleculeand standard molecule is routine (Johnson, Proteins 7:205-214, 1990).Crystallography is another well known method for analyzing folding andstructure. Nuclear magnetic resonance (NMR), digestive peptide mappingand epitope mapping are also known methods for analyzing folding andstructurally similarities between proteins and polypeptides (Schaanan etal., Science 257:961-964, 1992).

A Hopp/Woods hydrophilicity profile of the Zcyto21 protein sequence asshown in SEQ ID NO:2 can be generated (Hopp et al., Proc. Natl. Acad.Sci. 78:3824-3828, 1981; Hopp, J. Immun. Meth. 88:1-18, 1986 andTriquier et al., Protein Engineering 11:153-169, 1998). The profile isbased on a sliding six-residue window. Buried G, S, and T residues andexposed H, Y, and W residues were ignored. For example, in Zcyto21,hydrophilic regions include residues 155 (Glu) to 160 (Glu); residues 51(Lys) to 56 (Ala); residues 50 (Phe) to 55 (Asp); residues 140 (Pro) to145 (Arg); and residues 154 (Gln) to 159 (Lys); as shown in SEQ ID NO:2.

Those skilled in the art will recognize that hydrophilicity orhydrophobicity will be taken into account when designing modificationsin the amino acid sequence of a Zcyto21 polypeptide, so as not todisrupt the overall structural and biological profile. Of particularinterest for replacement are hydrophobic residues selected from thegroup consisting of Val, Leu and Ile or the group consisting of Met,Gly, Ser, Ala, Tyr and Trp.

The identities of essential amino acids can also be inferred fromanalysis of sequence similarity between INF-α and other interferons.Using methods such as “FASTA” analysis described previously, regions ofhigh similarity are identified within a family of proteins and used toanalyze amino acid sequence for conserved regions. An alternativeapproach to identifying a variant Zcyto21 polynucleotide on the basis ofstructure is to determine whether a nucleic acid molecule encoding apotential variant Zcyto21 gene can hybridize to a nucleic acid moleculehaving the nucleotide sequence of SEQ ID NO:1, as discussed above.

Other methods of identifying essential amino acids in the polypeptidesof the present invention are procedures known in the art, such assite-directed mutagenesis or alanine-scanning mutagenesis (Cunninghamand Wells, Science 244:1081 (1989), Bass et al., Proc. Natl. Acad. Sci.USA 88:4498 (1991), Coombs and Corey, “Site-Directed Mutagenesis andProtein Engineering,” in Proteins: Analysis and Design, Angeletti (ed.),pages 259-311 (Academic Press, Inc. 1998)). In the latter technique,single alanine mutations are introduced at every residue in themolecule, and the resultant mutant molecules are tested for biologicalor biochemical activity as disclosed below to identify amino acidresidues that are critical to the activity of the molecule. See also,Hilton et al, J. Biol. Chem. 271:4699 (1996).

The present invention also includes functional fragments of Zcyto21polypeptides and nucleic acid molecules encoding such functionalfragments. A “functional” Zcyto21 or fragment thereof as defined hereinis characterized by its proliferative or differentiating activity, byits ability to induce or inhibit specialized cell functions, or by itsability to bind specifically to an anti-Zcyto21 antibody or Zcyto21receptor (either soluble or immobilized). As previously describedherein, Zcyto21 is characterized by a six-helical-bundle structurecomprising: helix A is defined by amino acid residues 49 (Ser) to 63(Leu); helix B by amino acid residues 76 (Asn) to 84 (Val); helix C byamino acid residues 89 (Val) to 104 (Ala); helix D by amino acidresidues 111 (Glu) to 133 (Gln); helix E by amino acid residues 137(Thr) to 158 (Lys); and helix F by amino acid residues 163 (Gly) to 189(Leu); as shown in SEQ ID NO: 2. Thus, the present invention furtherprovides fusion proteins encompassing: (a) polypeptide moleculescomprising one or more of the helices described above; and (b)functional fragments comprising one or more of these helices. The otherpolypeptide portion of the fusion protein may be contributed by anotherhelical-bundle cytokine or interferon, such as INF-α, or by a non-nativeand/or an unrelated secretory signal peptide that facilitates secretionof the fusion protein.

The Zcyto21 polypeptides of the present invention, including full-lengthpolypeptides, biologically active fragments, and fusion polypeptides canbe produced according to conventional techniques using cells into whichhave been introduced an expression vector encoding the polypeptide. Asused herein, “cells into which have been introduced an expressionvector” include both cells that have been directly manipulated by theintroduction of exogenous DNA molecules and progeny thereof that containthe introduced DNA. Suitable host cells are those cell types that can betransformed or transfected with exogenous DNA and grown in culture, andinclude bacteria, fungal cells, and cultured higher eukaryotic cells.Techniques for manipulating cloned DNA molecules and introducingexogenous DNA into a variety of host cells are disclosed by Sambrook etal., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989, and Ausubel et al.,eds., Current Protocols in Molecular Biology, John Wiley and Sons, Inc.,NY, 1987.

In general, a DNA sequence encoding a Zcyto21 polypeptide is operablylinked to other genetic elements required for its expression, generallyincluding a transcription promoter and terminator, within an expressionvector. The vector will also commonly contain one or more selectablemarkers and one or more origins of replication, although those skilledin the art will recognize that within certain systems selectable markersmay be provided on separate vectors, and replication of the exogenousDNA may be provided by integration into the host cell genome. Selectionof promoters, terminators, selectable markers, vectors and otherelements is a matter of routine design within the level of ordinaryskill in the art. Many such elements are described in the literature andare available through commercial suppliers.

To direct a Zcyto21 polypeptide into the secretory pathway of a hostcell, a secretory signal sequence (also known as a leader sequence,prepro sequence or pre sequence) is provided in the expression vector.The secretory signal sequence may be that of Zcyto21, or may be derivedfrom another secreted protein (e.g., t-PA; see, U.S. Pat. No. 5,641,655)or synthesized de novo. The secretory signal sequence is operably linkedto the Zcyto21 DNA sequence, i.e., the two sequences are joined in thecorrect reading frame and positioned to direct the newly synthesizedpolypeptide into the secretory pathway of the host cell. Secretorysignal sequences are commonly positioned 5′ to the DNA sequence encodingthe polypeptide of interest, although certain signal sequences may bepositioned elsewhere in the DNA sequence of interest (see, e.g., Welchet al., U.S. Pat. No. 5,037,743; Holland et al., U.S. Pat. No.5,143,830).

Cultured mammalian cells can be used as hosts within the presentinvention. Methods for introducing exogenous DNA into mammalian hostcells include calcium phosphate-mediated transfection (Wigler et al.,Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603,1981; Graham and Van der Eb, Virology 52:456, 1973), electroporation(Neumann et al., EMBO J. 1:841-845, 1982), DEAE-dextran mediatedtransfection (Ausubel et al., ibid.), and liposome-mediated transfection(Hawley-Nelson et al., Focus 15:73, 1993; Ciccarone et al., Focus 15:80,1993). The production of recombinant polypeptides in cultured mammaliancells is disclosed, for example, by Levinson et al., U.S. Pat. No.4,713,339; Hagen et al., U.S. Pat. No. 4,784,950; Palmiter et al, U.S.Pat. No. 4,579,821; and Ringold, U.S. Pat. No. 4,656,134. Suitablecultured mammalian cells include the COS-1 (ATCC No. CRL 1650), COS-7(ATCC No. CRL 1651), BHK (ATCC No. CRL 1632), BHK 570 (ATCC No. CRL10314), 293 (ATCC No. CRL 1573; Graham et al., J. Gen. Virol. 36:59-72,1977) and Chinese hamster ovary (e.g. CHO-K1, ATCC No. CCL 61; or CHODG44, Chasin et al., Som. Cell. Molec. Genet. 12:555, 1986) cell lines.Additional suitable cell lines are known in the art and available frompublic depositories such as the American Type Culture Collection,Manassas, Va. In general, strong transcription promoters are preferred,such as promoters from SV-40 or cytomegalovirus. See, e.g., U.S. Pat.No. 4,956,288. Other suitable promoters include those frommetallothionein genes (U.S. Pat. Nos. 4,579,821 and 4,601,978) and theadenovirus major late promoter. Expression vectors for use in mammaliancells include pZP-1 and pZP-9, which have been deposited with theAmerican Type Culture Collection, Manassas, Va. USA under accessionnumbers 98669 and 98668, respectively, and derivatives thereof.

Drug selection is generally used to select for cultured mammalian cellsinto which foreign DNA has been inserted. Such cells are commonlyreferred to as “transfectants”. Cells that have been cultured in thepresence of the selective agent and are able to pass the gene ofinterest to their progeny are referred to as “stable transfectants.” Apreferred selectable marker is a gene encoding resistance to theantibiotic neomycin. Selection is carried out in the presence of aneomycin-type drug, such as G-418 or the like. Selection systems canalso be used to increase the expression level of the gene of interest, aprocess referred to as “amplification.” Amplification is carried out byculturing transfectants in the presence of a low level of the selectiveagent and then increasing the amount of selective agent to select forcells that produce high levels of the products of the introduced genes.A preferred amplifiable selectable marker is dihydrofolate reductase,which confers resistance to methotrexate. Other drug resistance genes(e.g. hygromycin resistance, multi-drug resistance, puromycinacetyltransferase) can also be used.

The adenovirus system can also be used for protein production in vitro.By culturing adenovirus-infected non-293 cells under conditions wherethe cells are not rapidly dividing, the cells can produce proteins forextended periods of time. For instance, BHK cells are grown toconfluence in cell factories, then exposed to the adenoviral vectorencoding the secreted protein of interest. The cells are then grownunder serum-free conditions, which allows infected cells to survive forseveral weeks without significant cell division. In an alternativemethod, adenovirus vector-infected 293 cells can be grown as adherentcells or in suspension culture at relatively high cell density toproduce significant amounts of protein (See Garnier et al., Cytotechnol.15:145-55, 1994). With either protocol, an expressed, secretedheterologous protein can be repeatedly isolated from the cell culturesupernatant, lysate, or membrane fractions depending on the dispositionof the expressed protein in the cell. Within the infected 293 cellproduction protocol, non-secreted proteins can also be effectivelyobtained.

Insect cells can be infected with recombinant baculovirus, commonlyderived from Autographa californica nuclear polyhedrosis virus (AcNPV)according to methods known in the art. Within a preferred method,recombinant baculovirus is produced through the use of atransposon-based system described by Luckow et al. (J. Virol.67:4566-4579, 1993). This system, which utilizes transfer vectors, iscommercially available in kit form (Bac-to-Bac™ kit; Life Technologies,Rockville, Md.). The transfer vector (e.g., pFastBac1™; LifeTechnologies) contains a Tn7 transposon to move the DNA encoding theprotein of interest into a baculovirus genome maintained in E. coli as alarge plasmid called a “bacmid.” See, Hill-Perkins and Possee, J. Gen.Virol. 71:971-976, 1990; Bonning et al., J. Gen. Virol. 75:1551-1556,1994; and Chazenbalk and Rapoport, J. Biol. Chem. 270:1543-1549, 1995.In addition, transfer vectors can include an in-frame fusion with DNAencoding a polypeptide extension or affinity tag as disclosed above.Using techniques known in the art, a transfer vector containing aZcyto21-encoding sequence is transformed into E. coli host cells, andthe cells are screened for bacmids which contain an interrupted lacZgene indicative of recombinant baculovirus. The bacmid DNA containingthe recombinant baculovirus genome is isolated, using common techniques,and used to transfect Spodoptera frugiperda cells, such as Sf9 cells.Recombinant virus that expresses Zcyto21 protein is subsequentlyproduced. Recombinant viral stocks are made by methods commonly used theart.

For protein production, the recombinant virus is used to infect hostcells, typically a cell line derived from the fall armyworm, Spodopterafrugiperda (e.g., Sf9 or Sf21 cells) or Trichoplusia ni (e.g., HighFive™ cells; Invitrogen, Carlsbad, Calif.). See, for example, U.S. Pat.No. 5,300,435. Serum-free media are used to grow and maintain the cells.Suitable media formulations are known in the art and can be obtainedfrom commercial suppliers. The cells are grown up from an inoculationdensity of approximately 2-5×10⁵ cells to a density of 1-2×10⁶ cells, atwhich time a recombinant viral stock is added at a multiplicity ofinfection (MOI) of 0.1 to 10, more typically near 3. Procedures used aregenerally known in the art.

Other higher eukaryotic cells can also be used as hosts, including plantcells and avian cells. The use of Agrobacterium rhizogenes as a vectorfor expressing genes in plant cells has been reviewed by Sinkar et al.,J. Biosci. (Bangalore) 11:47-58, 1987.

Fungal cells, including yeast cells, can also be used within the presentinvention. Yeast species of particular interest in this regard includeSaccharomyces cerevisiae, Pichia pastoris, and Pichia methanolica.Methods for transforming S. cerevisiae cells with exogenous DNA andproducing recombinant polypeptides therefrom are disclosed by, forexample, Kawasaki, U.S. Pat. No. 4,599,311; Kawasaki et al., U.S. Pat.No. 4,931,373; Brake, U.S. Pat. No. 4,870,008; Welch et al, U.S. Pat.No. 5,037,743; and Murray et al., U.S. Pat. No. 4,845,075. Transformedcells are selected by phenotype determined by the selectable marker,commonly drug resistance or the ability to grow in the absence of aparticular nutrient (e.g., leucine). A preferred vector system for usein Saccharomyces cerevisiae is the POT1 vector system disclosed byKawasaki et al. (U.S. Pat. No. 4,931,373), which allows transformedcells to be selected by growth in glucose-containing media. Suitablepromoters and terminators for use in yeast include those from glycolyticenzyme genes (see, e.g., Kawasaki, U.S. Pat. No. 4,599,311; Kingsman etal., U.S. Pat. No. 4,615,974; and Bitter, U.S. Pat. No. 4,977,092) andalcohol dehydrogenase genes. See also U.S. Pat. Nos. 4,990,446;5,063,154; 5,139,936 and 4,661,454. Transformation systems for otheryeasts, including Hansenula polymorpha, Schizosaccharomyces pombe,Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichiapastoris, Pichia methanolica, Pichia guillermondii and Candida maltosaare known in the art. See, for example, Gleeson et al., J. Gen.Microbiol. 132:3459-3465, 1986; Cregg, U.S. Pat. No. 4,882,279; andRaymond et al., Yeast 14, 11-23, 1998. Aspergillus cells may be utilizedaccording to the methods of McKnight et al., U.S. Pat. No. 4,935,349.Methods for transforming Acremonium chrysogenum are disclosed by Suminoet al., U.S. Pat. No. 5,162,228. Methods for transforming Neurospora aredisclosed by Lambowitz, U.S. Pat. No. 4,486,533. Production ofrecombinant proteins in Pichia methanolica is disclosed in U.S. Pat.Nos. 5,716,808, 5,736,383, 5,854,039, and 5,888,768.

Prokaryotic host cells, including strains of the bacteria Escherichiacoli, Bacillus and other genera are also useful host cells within thepresent invention. Techniques for transforming these hosts andexpressing foreign DNA sequences cloned therein are well known in theart (see, e.g., Sambrook et al., ibid.). When expressing a Zcyto21polypeptide in bacteria such as E. coli, the polypeptide may be retainedin the cytoplasm, typically as insoluble granules, or may be directed tothe periplasmic space by a bacterial secretion sequence. In the formercase, the cells are lysed, and the granules are recovered and denaturedusing, for example, guanidine isothiocyanate or urea. The denaturedpolypeptide can then be refolded and dimerized by diluting thedenaturant, such as by dialysis against a solution of urea and acombination of reduced and oxidized glutathione, followed by dialysisagainst a buffered saline solution. In the latter case, the polypeptidecan be recovered from the periplasmic space in a soluble and functionalform by disrupting the cells (by, for example, sonication or osmoticshock) to release the contents of the periplasmic space and recoveringthe protein, thereby obviating the need for denaturation and refolding.

Transformed or transfected host cells are cultured according toconventional procedures in a culture medium containing nutrients andother components required for the growth of the chosen host cells. Avariety of suitable media, including defined media and complex media,are known in the art and generally include a carbon source, a nitrogensource, essential amino acids, vitamins and minerals. Media may alsocontain such components as growth factors or serum, as required. Thegrowth medium will generally select for cells containing the exogenouslyadded DNA by, for example, drug selection or deficiency in an essentialnutrient which is complemented by the selectable marker carried on theexpression vector or co-transfected into the host cell. Liquid culturesare provided with sufficient aeration by conventional means, such asshaking of small flasks or sparging of fermentors.

It is preferred to purify the polypeptides and proteins of the presentinvention to ≧80% purity, more preferably to ≧90% purity, even morepreferably ≧95% purity, and particularly preferred is a pharmaceuticallypure state, that is greater than 99.9% pure with respect tocontaminating macromolecules, particularly other proteins and nucleicacids, and free of infectious and pyrogenic agents. Preferably, apurified polypeptide or protein is substantially free of otherpolypeptides or proteins, particularly those of animal origin.

Expressed recombinant Zcyto21 proteins (including chimeric polypeptidesand multimeric proteins) are purified by conventional proteinpurification methods, typically by a combination of chromatographictechniques. See, in general, Affinity Chromatography: Principles &Methods, Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988; and Scopes,Protein Purification: Principles and Practice, Springer-Verlag, NewYork, 1994. Proteins comprising a polyhistidine affinity tag (typicallyabout 6 histidine residues) are purified by affinity chromatography on anickel chelate resin. See, for example, Houchuli et al., Bio/Technol. 6:1321-1325, 1988. Proteins comprising a glu-glu tag can be purified byimmunoaffinity chromatography according to conventional procedures. See,for example, Grussenmeyer et al., ibid. Maltose binding protein fusionsare purified on an amylose column according to methods known in the art.

Zcyto21 polypeptides can also be prepared through chemical synthesisaccording to methods known in the art, including exclusive solid phasesynthesis, partial solid phase methods, fragment condensation orclassical solution synthesis. See, for example, Merrifield, J. Am. Chem.Soc. 85:2149, 1963; Stewart et al., Solid Phase Peptide Synthesis (2ndedition), Pierce Chemical Co., Rockford, Ill., 1984; Bayer and Rapp,Chem. Pept. Prot. 3:3, 1986; and Atherton et al., Solid Phase PeptideSynthesis: A Practical Approach, IRL Press, Oxford, 1989. In vitrosynthesis is particularly advantageous for the preparation of smallerpolypeptides.

Using methods known in the art, Zcyto21 proteins can be prepared asmonomers or multimers; glycosylated or non-glycosylated; pegylated ornon-pegylated; and may or may not include an initial methionine aminoacid residue.

Target cells for use in Zcyto21 activity assays include, withoutlimitation, vascular cells (especially endothelial cells and smoothmuscle cells), hematopoietic (myeloid and lymphoid) cells, liver cells(including hepatocytes, fenestrated endothelial cells, Kupffer cells,and Ito cells), fibroblasts (including human dermal fibroblasts and lungfibroblasts), fetal lung cells, articular synoviocytes, pericytes,chondrocytes, osteoblasts, and prostate epithelial cells. Endothelialcells and hematopoietic cells are derived from a common ancestral cell,the hemangioblast (Choi et al., Development 125:725-732, 1998).

Zcyto21 proteins of the present invention are characterized by theiractivity, that is, modulation of the proliferation, differentiation,migration, adhesion, or metabolism of responsive cell types. Biologicalactivity of Zcyto21 proteins is assayed using in vitro or in vivo assaysdesigned to detect cell proliferation, differentiation, migration oradhesion; or changes in cellular metabolism (e.g., production of othergrowth factors or other macromolecules). Many suitable assays are knownin the art, and representative assays are disclosed herein. Assays usingcultured cells are most convenient for screening, such as fordetermining the effects of amino acid substitutions, deletions, orinsertions. However, in view of the complexity of developmentalprocesses (e.g., angiogenesis, wound healing), in vivo assays willgenerally be employed to confirm and further characterize biologicalactivity. Certain in vitro models, such as the three-dimensionalcollagen gel matrix model of Pepper et al. (Biochem. Biophys. Res. Comm.189:824-831, 1992), are sufficiently complex to assay histologicaleffects. Assays can be performed using exogenously produced proteins, ormay be carried out in vivo or in vitro using cells expressing thepolypeptide(s) of interest. Assays can be conducted using Zcyto21proteins alone or in combination with other growth factors, such asmembers of the VEGF family or hematopoietic cytokines (e.g., EPO, TPO,G-CSF, stem cell factor). Representative assays are disclosed below.

Activity of Zcyto21 proteins can be measured in vitro using culturedcells or in vivo by administering molecules of the claimed invention toan appropriate animal model. Assays measuring cell proliferation ordifferentiation are well known in the art. For example, assays measuringproliferation include such assays as chemosensitivity to neutral red dye(Cavanaugh et al., Investigational New Drugs 8:347-354, 1990),incorporation of radiolabelled nucleotides (as disclosed by, e.g.,Raines and Ross, Methods Enzymol. 109:749-773, 1985; Wahl et al., Mol.Cell. Biol. 8:5016-5025, 1988; and Cook et al., Analytical Biochem.179:1-7, 1989), incorporation of 5-bromo-2′-deoxyuridine (BrdU) in theDNA of proliferating cells (Porstmann et al., J. Immunol. Methods82:169-179, 1985), and use of tetrazolium salts (Mosmann, J. Immunol.Methods 65:55-63, 1983; Alley et al., Cancer Res. 48:589-601, 1988;Marshall et al., Growth Reg. 5:69-84, 1995; and Scudiero et al., CancerRes. 48:4827-4833, 1988). Differentiation can be assayed using suitableprecursor cells that can be induced to differentiate into a more maturephenotype. Assays measuring differentiation include, for example,measuring cell-surface markers associated with stage-specific expressionof a tissue, enzymatic activity, functional activity or morphologicalchanges (Watt, FASEB, 5:281-284, 1991; Francis, Differentiation57:63-75, 1994; Raes, Adv. Anim. Cell Biol. Technol. Bioprocesses,161-171, 1989; all incorporated herein by reference).

Zcyto21 activity may also be detected using assays designed to measureZcyto21-induced production of one or more additional growth factors orother macromolecules. Preferred such assays include those fordetermining the presence of hepatocyte growth factor (HGF), epidermalgrowth factor (EGF), transforming growth factor alpha (TGFα),interleukin-6 (IL-6), VEGF, acidic fibroblast growth factor (aFGF),angiogenin, and other macromolecules produced by the liver. Suitableassays include mitogenesis assays using target cells responsive to themacromolecule of interest, receptor-binding assays, competition bindingassays, immunological assays (e.g., ELISA), and other formats known inthe art. Metalloprotease secretion is measured from treated primaryhuman dermal fibroblasts, synoviocytes and chondrocytes. The relativelevels of collagenase, gelatinase and stromalysin produced in responseto culturing in the presence of a Zcyto21 protein is measured usingzymogram gels (Loita and Stetler-Stevenson, Cancer Biology 1:96-106,1990). Procollagen/collagen synthesis by dermal fibroblasts andchondrocytes in response to a test protein is measured using ³H-prolineincorporation into nascent secreted collagen. ³H-labeled collagen isvisualized by SDS-PAGE followed by autoradiography (Unemori and Amento,J. Biol. Chem. 265: 10681-10685, 1990). Glycosaminoglycan (GAG)secretion from dermal fibroblasts and chondrocytes is measured using a1,9-dimethylmethylene blue dye binding assay (Farndale et al., Biochim.Biophys. Acta 883:173-177, 1986). Collagen and GAG assays are alsocarried out in the presence of IL-1α or TGF-α to examine the ability ofZcyto21 protein to modify the established responses to these cytokines.

Monocyte activation assays are carried out (1) to look for the abilityof Zcyto21 proteins to further stimulate monocyte activation, and (2) toexamine the ability of Zcyto21 proteins to modulate attachment-inducedor endotoxin-induced monocyte activation (Fuhlbrigge et al., J. Immunol.138: 3799-3802, 1987). IL-1α and TNFα levels produced in response toactivation are measured by ELISA (Biosource, Inc. Camarillo, Calif.).Monocyte/macrophage cells, by virtue of CD14 (LPS receptor), areexquisitely sensitive to endotoxin, and proteins with moderate levels ofendotoxin-like activity will activate these cells.

Hematopoietic activity of Zcyto21 proteins can be assayed on varioushematopoietic cells in culture. Preferred assays include primary bonemarrow colony assays and later stage lineage-restricted colony assays,which are known in the art (e.g., Holly et al., WIPO Publication WO95/21920). Marrow cells plated on a suitable semi-solid medium (e.g.,50% methylcellulose containing 15% fetal bovine serum, 10% bovine serumalbumin, and 0.6% PSN antibiotic mix) are incubated in the presence oftest polypeptide, then examined microscopically for colony formation.Known hematopoietic factors are used as controls. Mitogenic activity ofZcyto21 polypeptides on hematopoietic cell lines can be measured asdisclosed above.

Cell migration is assayed essentially as disclosed by Kahler et al.(Arteriosclerosis Thrombosis, and Vascular Biology 17:932-939, 1997). Aprotein is considered to be chemotactic if it induces migration of cellsfrom an area of low protein concentration to an area of high proteinconcentration. A typical assay is performed using modified Boydenchambers with a polystryrene membrane separating the two chambers(Transwell; Corning Costar Corp.). The test sample, diluted in mediumcontaining 1% BSA, is added to the lower chamber of a 24-well platecontaining Transwells. Cells are then placed on the Transwell insertthat has been pretreated with 0.2% gelatin. Cell migration is measuredafter 4 hours of incubation at 37° C. Non-migrating cells are wiped offthe top of the Transwell membrane, and cells attached to the lower faceof the membrane are fixed and stained with 0.1% crystal violet. Stainedcells are then extracted with 10% acetic acid and absorbance is measuredat 600 nm. Migration is then calculated from a standard calibrationcurve. Cell migration can also be measured using the matrigel method ofGrant et al. (“Angiogenesis as a component of epithelial-mesenchymalinteractions” in Goldberg and Rosen, Epithelial-Mesenchymal Interactionin Cancer, Birkhäuser Verlag, 1995, 235-248; Baatout, AnticancerResearch 17:451-456, 1997).

Cell adhesion activity is assayed essentially as disclosed by LaFleur etal. (J. Biol. Chem. 272:32798-32803, 1997). Briefly, microtiter platesare coated with the test protein, non-specific sites are blocked withBSA, and cells (such as smooth muscle cells, leukocytes, or endothelialcells) are plated at a density of approximately 10⁴-10⁵ cells/well. Thewells are incubated at 37° C. (typically for about 60 minutes), thennon-adherent cells are removed by gentle washing. Adhered cells arequantitated by conventional methods (e.g., by staining with crystalviolet, lysing the cells, and determining the optical density of thelysate). Control wells are coated with a known adhesive protein, such asfibronectin or vitronectin.

The activity of Zcyto21 proteins can be measured with a silicon-basedbiosensor microphysiometer that measures the extracellular acidificationrate or proton excretion associated with receptor binding and subsequentphysiologic cellular responses. An exemplary such device is theCytosensor™ Microphysiometer manufactured by Molecular Devices,Sunnyvale, Calif. A variety of cellular responses, such as cellproliferation, ion transport, energy production, inflammatory response,regulatory and receptor activation, and the like, can be measured bythis method. See, for example, McConnell et al., Science 257:1906-1912,1992; Pitchford et al., Meth. Enzymol. 228:84-108, 1997; Arimilli etal., J. Immunol. Meth. 212:49-59, 1998; and Van Liefde et al., Eur. J.Pharmacol. 346:87-95, 1998. The microphysiometer can be used forassaying adherent or non-adherent eukaryotic or prokaryotic cells. Bymeasuring extracellular acidification changes in cell media over time,the microphysiometer directly measures cellular responses to variousstimuli, including Zcyto21 proteins, their agonists, and antagonists.Preferably, the microphysiometer is used to measure responses of aZcyto21-responsive eukaryotic cell, compared to a control eukaryoticcell that does not respond to Zcyto21 polypeptide. Zcyto21-responsiveeukaryotic cells comprise cells into which a receptor for Zcyto21 hasbeen transfected, thereby creating a cell that is responsive to Zcyto21,as well as cells naturally responsive to Zcyto21. Differences, measuredby a change, for example, an increase or diminution in extracellularacidification, in the response of cells exposed to Zcyto21 polypeptide,relative to a control not exposed to Zcyto21, are a direct measurementof Zcyto21-modulated cellular responses. Moreover, suchZcyto21-modulated responses can be assayed under a variety of stimuli.The present invention thus provides methods of identifying agonists andantagonists of Zcyto21 proteins, comprising providing cells responsiveto a Zcyto21 polypeptide, culturing a first portion of the cells in theabsence of a test compound, culturing a second portion of the cells inthe presence of a test compound, and detecting a change, for example, anincrease or diminution, in a cellular response of the second portion ofthe cells as compared to the first portion of the cells. The change incellular response is shown as a measurable change in extracellularacidification rate. Culturing a third portion of the cells in thepresence of a Zcyto21 protein and the absence of a test compoundprovides a positive control for the Zcyto21-responsive cells and acontrol to compare the agonist activity of a test compound with that ofthe Zcyto21 polypeptide. Antagonists of Zcyto21 can be identified byexposing the cells to Zcyto21 protein in the presence and absence of thetest compound, whereby a reduction in Zcyto21-stimulated activity isindicative of antagonist activity in the test compound.

Expression of Zcyto21 polynucleotides in animals provides models forfurther study of the biological effects of overproduction or inhibitionof protein activity in vivo. Zcyto21-encoding polynucleotides andantisense polynucleotides can be introduced into test animals, such asmice, using viral vectors or naked DNA, or transgenic animals can beproduced.

One in vivo approach for assaying proteins of the present inventionutilizes viral delivery systems. Exemplary viruses for this purposeinclude adenovirus, herpesvirus, retroviruses, vaccinia virus, andadeno-associated virus (AAV). Adenovirus, a double-stranded DNA virus,is currently the best studied gene transfer vector for delivery ofheterologous nucleic acids. For review, see Becker et al., Meth. CellBiol. 43:161-89, 1994; and Douglas and Curiel, Science & Medicine4:44-53, 1997. The adenovirus system offers several advantages.Adenovirus can (i) accommodate relatively large DNA inserts; (ii) begrown to high-titer; (iii) infect a broad range of mammalian cell types;and (iv) be used with many different promoters including ubiquitous,tissue specific, and regulatable promoters. Because adenoviruses arestable in the bloodstream, they can be administered by intravenousinjection.

By deleting portions of the adenovirus genome, larger inserts (up to 7kb) of heterologous DNA can be accommodated. These inserts can beincorporated into the viral DNA by direct ligation or by homologousrecombination with a co-transfected plasmid. In an exemplary system, theessential E1 gene is deleted from the viral vector, and the virus willnot replicate unless the E1 gene is provided by the host cell (e.g., thehuman 293 cell line). When intravenously administered to intact animals,adenovirus primarily targets the liver. If the adenoviral deliverysystem has an E1 gene deletion, the virus cannot replicate in the hostcells. However, the host's tissue (e.g., liver) will express and process(and, if a signal sequence is present, secrete) the heterologousprotein. Secreted proteins will enter the circulation in the highlyvascularized liver, and effects on the infected animal can bedetermined.

An alternative method of gene delivery comprises removing cells from thebody and introducing a vector into the cells as a naked DNA plasmid. Thetransformed cells are then re-implanted in the body. Naked DNA vectorsare introduced into host cells by methods known in the art, includingtransfection, electroporation, microinjection, transduction, cellfusion, DEAE dextran, calcium phosphate precipitation, use of a genegun, or use of a DNA vector transporter. See, Wu et al., J. Biol. Chem.263:14621-14624, 1988; Wu et al., J. Biol. Chem. 267:963-967, 1992; andJohnston and Tang, Meth. Cell Biol. 43:353-365, 1994.

Transgenic mice, engineered to express a Zcyto21 gene, and mice thatexhibit a complete absence of Zcyto21 gene function, referred to as“knockout mice” (Snouwaert et al., Science 257:1083, 1992), can also begenerated (Lowell et al., Nature 366:740-742, 1993). These mice can beemployed to study the Zcyto21 gene and the protein encoded thereby in anin vivo system. Transgenic mice are particularly useful forinvestigating the role of Zcyto21 proteins in early development in thatthey allow the identification of developmental abnormalities or blocksresulting from the over- or underexpression of a specific factor. Seealso, Maisonpierre et al., Science 277:55-60, 1997 and Hanahan, Science277:48-50, 1997. Preferred promoters for transgenic expression includepromoters from metallothionein and albumin genes.

A loss of normal inhibitory control of muscle contraction has beenassociated with damage or perturbation of selected gamma-aminobutryricacid-secreting neurons. For example, Stiff Man Syndrome exhibitremarkable stiffness of musculature, believed to be mediated throughinterference of the functioning of their gamma-aminobutryric acid (GABA)producing neurons. Other related neuromuscular disorders includemyotonia, metabolic myopathies, Isaac's syndrome, dystonia, and tetanicspasms (Valldeoriola, J. Neurol 246:423-431, 1999).

Similarly, direct measurement of Zcyto21 polypeptide, or its loss ofexpression in a tissue can be determined in a tissue or cells as theyundergo tumor progression. Increases in invasiveness and motility ofcells, or the gain or loss of expression of Zcyto21 in a pre-cancerousor cancerous condition, in comparison to normal tissue, can serve as adiagnostic for transformation, invasion and metastasis in tumorprogression. As such, knowledge of a tumor's stage of progression ormetastasis will aid the physician in choosing the most proper therapy,or aggressiveness of treatment, for a given individual cancer patient.Methods of measuring gain and loss of expression (of either mRNA orprotein) are well known in the art and described herein and can beapplied to Zcyto21 expression. For example, appearance or disappearanceof polypeptides that regulate cell motility can be used to aid diagnosisand prognosis of prostate cancer (Banyard, J. and Zetter, B. R., Cancerand Metast. Rev. 17:449-458, 1999). As an effector of cell motility, oras a liver-specific marker, Zcyto21 gain or loss of expression may serveas a diagnostic for brain and other cancers. Moreover, analogous to theprostate specific antigen (PSA), increased levels of Zcyto21polypeptides, or anti-Zcyto21 antibodies in a patient, relative to anormal control can be indicative of brain and other cancers (See, e.g.,Mulders, T M T, et al., Eur. J. Surgical Oncol. 16:37-41, 1990). StrongZcyto21 expression in tissue not normally found to express Zcyto21 wouldserve as a diagnostic of an abnormality in the cell or tissue type, ofinvasion or metastasis of cancerous liver tissue into non-liver tissue,and could aid a physician in directing further testing or investigation,or aid in directing therapy.

In addition, Zcyto21 polynucleotide probes, anti-Zcyto21 antibodies, anddetection the presence of Zcyto21 polypeptides in tissue can be used toassess whether brain or other tissue found to normally express Zcyto21is present, for example, after surgery involving the excision of adiseased or cancerous liver or neuronal tissue. As such, thepolynucleotides, polypeptides, and antibodies of the present inventioncan be used as an aid to determine whether all tissue is excised aftersurgery, for example, after surgery for brain and other cancers. In suchinstances, it is especially important to remove all potentially diseasedtissue to maximize recovery from the cancer, and to minimize recurrence.Preferred embodiments include fluorescent, radiolabeled, orcalorimetrically labeled anti-Zcyto21 antibodies and Zcyto21 polypeptidebinding partners, that can be used histologically or in situ.

Moreover, the activity and effect of Zcyto21 on tumor progression andmetastasis can be measured in vivo. Several syngeneic mouse models havebeen developed to study the influence of polypeptides, compounds orother treatments on tumor progression. In these models, tumor cellspassaged in culture are implanted into mice of the same strain as thetumor donor. The cells will develop into tumors having similarcharacteristics in the recipient mice, and metastasis will also occur insome of the models. Appropriate tumor models for our studies include theLewis lung carcinoma (ATCC No. CRL-1642) and B16 melanoma (ATCC No.CRL-6323), amongst others. These are both commonly used tumor lines,syngeneic to the C57BL6 mouse, that are readily cultured and manipulatedin vitro. Tumors resulting from implantation of either of these celllines are capable of metastasis to the lung in C57BL6 mice. The Lewislung carcinoma model has recently been used in mice to identify aninhibitor of angiogenesis (O'Reilly M S, et al. Cell 79: 315-328, 1994).C57BL6/J mice are treated with an experimental agent either throughdaily injection of recombinant protein, agonist or antagonist or aone-time injection of recombinant adenovirus. Three days following thistreatment, 10⁵ to 10⁶ cells are implanted under the dorsal skin.Alternatively, the cells themselves may be infected with recombinantadenovirus, such as one expressing Zcyto21, before implantation so thatthe protein is synthesized at the tumor site or intracellularly, ratherthan systemically. The mice normally develop visible tumors within 5days. The tumors are allowed to grow for a period of up to 3 weeks,during which time they may reach a size of 1500-1800 mm³ in the controltreated group. Tumor size and body weight are carefully monitoredthroughout the experiment. At the time of sacrifice, the tumor isremoved and weighed along with the lungs and the liver. The lung weighthas been shown to correlate well with metastatic tumor burden. As anadditional measure, lung surface metastases are counted. The resectedtumor, lungs and liver are prepared for histopathological examination,immunohistochemistry, and in situ hybridization, using methods known inthe art and described herein. The influence of the expressed polypeptidein question, e.g., Zcyto21, on the ability of the tumor to recruitvasculature and undergo metastasis can thus be assessed. In addition,aside from using adenovirus, the implanted cells can be transientlytransfected with Zcyto21. Use of stable Zcyto21 transfectants as well asuse of induceable promoters to activate Zcyto21 expression in vivo areknown in the art and can be used in this system to assess Zcyto21induction of metastasis. Moreover, purified Zcyto21 orZcyto21-conditioned media can be directly injected in to this mousemodel, and hence be used in this system. For general reference see,O'Reilly M S, et al. Cell 79:315-328, 1994; and Rusciano D, et al.Murine Models of Liver Metastasis. Invasion Metastasis 14:349-361, 1995.

Antisense methodology can be used to inhibit Zcyto21 gene transcriptionto examine the effects of such inhibition in vivo. Polynucleotides thatare complementary to a segment of a Zcyto21-encoding polynucleotide(e.g., a polynucleotide as set forth in SEQ ID NO:1) are designed tobind to Zcyto211-encoding mRNA and to inhibit translation of such mRNA.Such antisense oligonucleotides can also be used to inhibit expressionof Zcyto21 polypeptide-encoding genes in cell culture.

Most cytokines as well as other proteins produced by activatedlymphocytes play an important biological role in cell differentiation,activation, recruitment and homeostasis of cells throughout the body.Zcyto21 and inhibitors of Zcyto21 activity are expected to have avariety of therapeutic applications. These therapeutic applicationsinclude treatment of diseases which require immune regulation, includingautoimmune diseases such as rheumatoid arthritis, multiple sclerosis,myasthenia gravis, systemic lupus erythematosis, and diabetes. Zcyto21may be important in the regulation of inflammation, and therefore wouldbe useful in treating rheumatoid arthritis, asthma and sepsis. There maybe a role of Zcyto21 in mediating tumorgenesis, whereby a Zcyto21antagonist would be useful in the treatment of cancer. Zcyto21 may beuseful in modulating the immune system, whereby Zcyto21 and Zcyto21antagonists may be used for reducing graft rejection, preventinggraft-vs-host disease, boosting immunity to infectious diseases,treating immunocompromised patients (e.g., HIV⁺ patients), or inimproving vaccines.

As an interferon-like polypeptide in tissues of brain, islet, prostate,testis, pituitary, placenta, ovarian tumor, lung tumor, rectal tumor andovarian tumor, as well as a CD3+ cell line, and a virally infectedprostate epithelial cell line, Zcyto21 is useful to modulate viralinfection, tumorigeneses and metastatis in these and other tissues. Insuch cases, the interferon-like molecule can be released by cell at thesite of infection or abnormal cell growth, or as a secreted molecule, itcan migrate to the site from a distant tissue.

The antiviral properties of Zcyto21 are particularly useful in treatinginfection with papilloma viruses in vitro and in vivo. For example,tumors caused by human papilloma viruses cause benign tumors (i.e.,genital warts) as well as malignant tumors such as squamous-cellcarcinomas. Treatment for these conditions commonly is surgery or tissuedestruction. Currently, however, some antiviral/immunomodulatory drugs,including interferon alpha, have been shown effective in reduce tumorsize. See Baker, G. et al., Dermatol. Clin. Apr. 15: 331-340, 1997.Further, as discussed by Rockley, P. F. et al., (Pharmacol. Ther. 65(2):265-287, 1995), immunologic therapy with interferons can be directedagainst all sites of infection, including clinical, subclinical, andlatent disease. In this example, IFN-alpha, IFN-beta and IFN-gamma havebeen used successfully as monotherapy as well as in combination withother therapies to treat anogenital condyloma acuminatum. Zcyto21 willbe a useful treatment similar to IFN-alpha, IFN-beta and IFN-gamma inthis treatment. Further more, there has been a strong associationbetween certain types of human papilloma virus and cervical cancer.Zcyto21 can be used to detect, monitor and treat cervical cancers.

As a small, secreted protein in islet cells Zcyto21 can modulate thegrowth and differentiation of these cells. Additionally, Zcyto21 may beuseful in treating diabetes and immunological conditions related to thegrowth and differentiation of the cells.

The presence of Zcyto21 in brain and pituitary cells indicates that itmay also find use in growth and differentiation of these cells. Further,the molecules of the present invention may be responsible fornutritional homeostasis, including behavioral disorders related tofeeding and appetite suppression. Additionally, Zcyto21 molecules mayfind use in treating reproductive disorders in general.

Zcyto21 polypeptides can be administered alone or in combination withother vasculogenic or angiogenic agents, including VEGF. When usingZcyto21 in combination with an additional agent, the two compounds canbe administered simultaneously or sequentially as appropriate for thespecific condition being treated.

Zcyto21 will be useful in treating tumorgenesis, and therefore would beuseful in the treatment of cancer. A Zcyto21 inhibition of anti-IgMstimulated normal B-cells and a similar effect is observed in B-celltumor lines suggest that there may be therapeutic benefit in treatingpatients with the Zcyto21 in order to induce the B cell tumor cells intoa less proliferative state. The ligand could be administered incombination with other agents already in use including both conventionalchemotherapeutic agents as well as immune modulators such as interferonalpha. Alpha/beta interferons have been shown to be effective intreating some leukemias and animal disease models, and the growthinhibitory effects of interferon-alpha and Zcyto21 may be additive forB-cell tumor-derived cell lines.

The present invention provides a method of reducing proliferation of aneoplastic B or T cells comprising administering to a mammal with a B orT cell neoplasm an amount of a composition of Zcyto21 sufficient toreduce proliferation of the neoplastic B or T cells. Zcyto21 stimulationof lytic NK cells from marrow progenitors and the proliferation ofT-cells following activation of the antigen receptors would enhancetreatment for patients receiving allogenic marrow transplants, andtherefore, Zcyto21 will enhance the generation of anti-tumor responses,with or without the infusion of donor lymphocytes.

In another aspect, the present invention provides a method of reducingproliferation of a neoplastic B or T cells comprising administering to amammal with a B or T cell neoplasm an amount of a composition of Zcyto21antagonist sufficient to reducing proliferation of the neoplastic B or Tcells. Furthermore, the Zcyto21 antagonist can be a ligand/toxin fusionprotein.

A Zcyto21-saporin fusion toxin may be employed against a similar set ofleukemias and lymphomas, extending the range of leukemias that can betreated with Zcyto21. Fusion toxin mediated activation of the Zcyto21receptor provides two independent means to inhibit the growth of thetarget cells, the first being identical to the effects seen by theligand alone, and the second due to delivery of the toxin throughreceptor internalization.

Based on the teachings herein, the interferon-like Zcyto21 molecules ofthe present invention will be useful to detect, monitor or treat suchdiverse conditions as hairy cell leukemia, renal cell carcinoma, basalcell carcinoma, malignant melanoma, AIDS-related Kaposi's sarcoma,multiple myeloma, chronic myelogenous leukemia, non-Hodgkin's lymphoma,laryngeal papillomatosis, mycosis fungoides, condyloma acuminata,papillomavirus-induced epidermodysplasi verruciformis, chronic hepatitisB, hepatitis C, chronic hepatitis D, and chronic non-A, non-B/Chepatitis. The U.S. Food and Drug Administration has approved the use ofinterferon-β, to treat multiple sclerosis, a chronic disease of thenervous system. Interferon-γ is used to treat chronic granulomatousdiseases, in which the interferon enhances the patient's immune responseto destroy infectious bacterial, fungal, and protozoal pathogens.Clinical studies also indicate that interferon-γ may be useful in thetreatment of AIDS, leishmaniasis, and lepromatous leprosy

For pharmaceutical use, Zcyto21 proteins are formulated for topical orparenteral, particularly intravenous or subcutaneous, delivery accordingto conventional methods. In general, pharmaceutical formulations willinclude a Zcyto21 polypeptide in combination with a pharmaceuticallyacceptable vehicle, such as saline, buffered saline, 5% dextrose inwater, or the like. Formulations may further include one or moreexcipients, preservatives, solubilizers, buffering agents, albumin toprevent protein loss on vial surfaces, etc. Methods of formulation arewell known in the art and are disclosed, for example, in Remington: TheScience and Practice of Pharmacy, Gennaro, ed., Mack Publishing Co.,Easton, Pa., 19th ed., 1995. Zcyto21 will preferably be used in aconcentration of about 10 to 100 μg/ml of total volume, althoughconcentrations in the range of 1 ng/ml to 1000 μg/ml may be used. Fortopical application, such as for the promotion of wound healing, theprotein will be applied in the range of 0.1-10 μg/cm² of wound area,with the exact dose determined by the clinician according to acceptedstandards, taking into account the nature and severity of the conditionto be treated, patient traits, etc. Determination of dose is within thelevel of ordinary skill in the art. Dosing is daily or intermittentlyover the period of treatment. Intravenous administration will be bybolus injection or infusion over a typical period of one to severalhours. Sustained release formulations can also be employed. In general,a therapeutically effective amount of Zcyto21 is an amount sufficient toproduce a clinically significant change in the treated condition, suchas a clinically significant change in hematopoietic or immune function,a significant reduction in morbidity, or a significantly increasedhistological score.

Zcyto21 proteins, agonists, and antagonists are useful for modulatingthe expansion, proliferation, activation, differentiation, migration, ormetabolism of responsive cell types, which include both primary cellsand cultured cell lines. Of particular interest in this regard arehematopoietic cells, mesenchymal cells (including stem cells and maturemyeloid and lymphoid cells), endothelial cells, epithelial cells, smoothmuscle cells, fibroblasts, hepatocytes, neural cells and embryonic stemcells. Zcyto21 polypeptides are added to tissue culture media for thesecell types at a concentration of about 10 pg/ml to about 100 ng/ml.Those skilled in the art will recognize that Zcyto21 proteins can beadvantageously combined with other growth factors in culture media.

Within the laboratory research field, Zcyto21 proteins can also be usedas molecular weight standards or as reagents in assays for determiningcirculating levels of the protein, such as in the diagnosis of disorderscharacterized by over- or under-production of Zcyto21 protein or in theanalysis of cell phenotype.

Zcyto21 proteins can also be used to identify inhibitors of theiractivity. Test compounds are added to the assays disclosed above toidentify compounds that inhibit the activity of Zcyto21 protein. Inaddition to those assays disclosed above, samples can be tested forinhibition of Zcyto21 activity within a variety of assays designed tomeasure receptor binding or the stimulation/inhibition ofZcyto21-dependent cellular responses. For example, Zcyto21-responsivecell lines can be transfected with a reporter gene construct that isresponsive to a Zcyto21-stimulated cellular pathway. Reporter geneconstructs of this type are known in the art, and will generallycomprise a Zcyto21-activated serum response element (SRE) operablylinked to a gene encoding an assayable protein, such as luciferase.Candidate compounds, solutions, mixtures or extracts are tested for theability to inhibit the activity of Zcyto21 on the target cells asevidenced by a decrease in Zcyto21 stimulation of reporter geneexpression. Assays of this type will detect compounds that directlyblock Zcyto21 binding to cell-surface receptors, as well as compoundsthat block processes in the cellular pathway subsequent toreceptor-ligand binding. In the alternative, compounds or other samplescan be tested for direct blocking of Zcyto21 binding to receptor usingZcyto21 tagged with a detectable label (e.g., ¹²⁵I, biotin, horseradishperoxidase, FITC, and the like). Within assays of this type, the abilityof a test sample to inhibit the binding of labeled Zcyto21 to thereceptor is indicative of inhibitory activity, which can be confirmedthrough secondary assays. Receptors used within binding assays may becellular receptors or isolated, immobilized receptors.

As used herein, the term “antibodies” includes polyclonal antibodies,monoclonal antibodies, antigen-binding fragments thereof such as F(ab′)₂and Fab fragments, single chain antibodies, and the like, includinggenetically engineered antibodies. Non-human antibodies may be humanizedby grafting non-human CDRs onto human framework and constant regions, orby incorporating the entire non-human variable domains (optionally“cloaking” them with a human-like surface by replacement of exposedresidues, wherein the result is a “veneered” antibody). In someinstances, humanized antibodies may retain non-human residues within thehuman variable region framework domains to enhance proper bindingcharacteristics. Through humanizing antibodies, biological half-life maybe increased, and the potential for adverse immune reactions uponadministration to humans is reduced. One skilled in the art can generatehumanized antibodies with specific and different constant domains (i.e.,different Ig subclasses) to facilitate or inhibit various immunefunctions associated with particular antibody constant domains.Antibodies are defined to be specifically binding if they bind to aZcyto21 polypeptide or protein with an affinity at least 10-fold greaterthan the binding affinity to control (non-Zcyto21) polypeptide orprotein. The affinity of a monoclonal antibody can be readily determinedby one of ordinary skill in the art (see, for example, Scatchard, Ann.NY Acad. Sci. 51: 660-672, 1949).

Methods for preparing polyclonal and monoclonal antibodies are wellknown in the art (see for example, Hurrell, J. G. R., Ed., MonoclonalHybridoma Antibodies: Techniques and Applications, CRC Press, Inc., BocaRaton, Fla., 1982, which is incorporated herein by reference). Ofparticular interest are generating antibodies to hydrophilic antigenicsites which include, for example, residues 155 (Glu) to 160 (Glu);residues 51 (Lys) to 56 (Ala); residues 50 (Phe) to 55 (Asp); residues140 (Pro) to 145 (Arg); and residues 154 (Gln) to 159 (Lys); as shown inSEQ ID NO: 2. As would be evident to one of ordinary skill in the art,polyclonal antibodies can be generated from a variety of warm-bloodedanimals such as horses, cows, goats, sheep, dogs, chickens, rabbits,mice, and rats. The immunogenicity of a Zcyto21 polypeptide may beincreased through the use of an adjuvant such as alum (aluminumhydroxide) or Freund's complete or incomplete adjuvant. Polypeptidesuseful for immunization also include fusion polypeptides, such asfusions of a Zcyto21 polypeptide or a portion thereof with animmunoglobulin polypeptide or with maltose binding protein. Thepolypeptide immunogen may be a full-length molecule or a portionthereof. If the polypeptide portion is “hapten-like”, such portion maybe advantageously joined or linked to a macromolecular carrier (such askeyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or tetanustoxoid) for immunization.

Alternative techniques for generating or selecting antibodies include invitro exposure of lymphocytes to Zcyto21 polypeptides, and selection ofantibody display libraries in phage or similar vectors (e.g., throughthe use of immobilized or labeled Zcyto21 polypeptide). Human antibodiescan be produced in transgenic, non-human animals that have beenengineered to contain human immunoglobulin genes as disclosed in WIPOPublication WO 98/24893. It is preferred that the endogenousimmunoglobulin genes in these animals be inactivated or eliminated, suchas by homologous recombination.

A variety of assays known to those skilled in the art can be utilized todetect antibodies which specifically bind to Zcyto21 polypeptides.Exemplary assays are described in detail in Antibodies: A LaboratoryManual, Harlow and Lane (Eds.), Cold Spring Harbor Laboratory Press,1988. Representative examples of such assays include: concurrentimmunoelectrophoresis, radio-immunoassays, radio-immunoprecipitations,enzyme-linked immunosorbent assays (ELISA), dot blot assays, Westernblot assays, inhibition or competition assays, and sandwich assays.

Antibodies to Zcyto21 may be used for affinity purification of theprotein, within diagnostic assays for determining circulating levels ofthe protein; for detecting or quantitating soluble Zcyto21 polypeptideas a marker of underlying pathology or disease; for immunolocalizationwithin whole animals or tissue sections, including immunodiagnosticapplications; for immunohistochemistry; and as antagonists to blockprotein activity in vitro and in vivo. Antibodies to Zcyto21 may also beused for tagging cells that express Zcyto21; for affinity purificationof Zcyto21 polypeptides and proteins; in analytical methods employingFACS; for screening expression libraries; and for generatinganti-idiotypic antibodies. Antibodies can be linked to other compounds,including therapeutic and diagnostic agents, using known methods toprovide for targeting of those compounds to cells expressing receptorsfor Zcyto21. For certain applications, including in vitro and in vivodiagnostic uses, it is advantageous to employ labeled antibodies.Suitable direct tags or labels include radionuclides, enzymes,substrates, cofactors, inhibitors, fluorescent markers, chemiluminescentmarkers, magnetic particles and the like; indirect tags or labels mayfeature use of biotin-avidin or other complement/anti-complement pairsas intermediates. Antibodies of the present invention may also bedirectly or indirectly conjugated to drugs, toxins, radionuclides andthe like, and these conjugates used for in vivo diagnostic ortherapeutic applications (e.g., inhibition of cell proliferation). See,in general, Ramakrishnan et al., Cancer Res. 56:1324-1330, 1996.

Polypeptides and proteins of the present invention can be used toidentify and isolate receptors. Zcyto21 receptors may be involved ingrowth regulation in the liver, blood vessel formation, and otherdevelopmental processes. For example, Zcyto21 proteins and polypeptidescan be immobilized on a column, and membrane preparations run over thecolumn (as generally disclosed in Immobilized Affinity LigandTechniques, Hermanson et al., eds., Academic Press, San Diego, Calif.,1992, pp. 195-202). Proteins and polypeptides can also be radiolabeled(Methods Enzymol., vol. 182, “Guide to Protein Purification”, M.Deutscher, ed., Academic Press, San Diego, 1990, 721-737) orphotoaffinity labeled (Brunner et al., Ann. Rev. Biochem. 62:483-514,1993 and Fedan et al., Biochem. Pharmacol. 33:1167-1180, 1984) and usedto tag specific cell-surface proteins. In a similar manner, radiolabeledZcyto21 proteins and polypeptides can be used to clone the cognatereceptor in binding assays using cells transfected with an expressioncDNA library.

Zcyto21 polypeptides can also be used to teach analytical skills such asmass spectrometry, circular dichroism, to determine conformation,especially of the four alpha helices, x-ray crystallography to determinethe three-dimensional structure in atomic detail, nuclear magneticresonance spectroscopy to reveal the structure of proteins in solution.For example, a kit containing the Zcyto21 can be given to the student toanalyze. Since the amino acid sequence would be known by the instructor,the protein can be given to the student as a test to determine theskills or develop the skills of the student, the instructor would thenknow whether or not the student has correctly analyzed the polypeptide.Since every polypeptide is unique, the educational utility of Zcyto21would be unique unto itself.

The antibodies which bind specifically to Zcyto21 can be used as ateaching aid to instruct students how to prepare affinity chromatographycolumns to purify Zcyto21, cloning and sequencing the polynucleotidethat encodes an antibody and thus as a practicum for teaching a studenthow to design humanized antibodies. The Zcyto21 gene, polypeptide, orantibody would then be packaged by reagent companies and sold toeducational institutions so that the students gain skill in art ofmolecular biology. Because each gene and protein is unique, each geneand protein creates unique challenges and learning experiences forstudents in a lab practicum. Such educational kits containing theZcyto21 gene, polypeptide, or antibody are considered within the scopeof the present invention. The present invention, thus generallydescribed, will be understood more readily by reference to the followingexamples, which is provided by way of illustration and is not intendedto be limiting of the present invention.

EXAMPLES Example 1

An expression plasmid containing all or part of a polynucleotideencoding Zcyto21 is constructed via homologous recombination. A fragmentof Zcyto21 cDNA is isolated by PCR using the polynucleotide sequence ofSEQ ID NO:1 with flanking regions at the 5′ and 3′ ends corresponding tothe vector sequences flanking the Zcyto21 insertion point. The primersfor PCR each include from 5′ to 3′ end: 40 bp of flanking sequence fromthe vector and 17 bp corresponding to the amino and carboxyl terminifrom the open reading frame of Zcyto21.

Ten μl of the 100 μl PCR reaction mixture is run on a 0.8%low-melting-temperature agarose (SeaPlaque GTG®; FMC BioProducts,Rockland, Me.) gel with 1×TBE buffer for analysis. The remaining 90 μlof the reaction mixture is precipitated with the addition of 5 μl 1 MNaCl and 250 μl of absolute ethanol. The plasmid pZMP6, which has beencut with SmaI, is used for recombination with the PCR fragment. PlasmidpZMP6 is a mammalian expression vector containing an expression cassettehaving the cytomegalovirus immediate early promoter, multiplerestriction sites for insertion of coding sequences, a stop codon, and ahuman growth hormone terminator; an E. coli origin of replication; amammalian selectable marker expression unit comprising an SV40 promoter,enhancer and origin of replication, a DHFR gene, and the SV40terminator; and URA3 and CEN-ARS sequences required for selection andreplication in S. cerevisiae. It was constructed from pZP9 (deposited atthe American Type Culture Collection, 10801 University Boulevard,Manassas, Va. 20110-2209, under Accession No. 98668) with the yeastgenetic elements taken from pRS316 (deposited at the American TypeCulture Collection, 10801 University Boulevard, Manassas, Va.20110-2209, under Accession No. 77145), an internal ribosome entry site(IRES) element from poliovirus, and the extracellular domain of CD8truncated at the C-terminal end of the transmembrane domain.

One hundred microliters of competent yeast (S. cerevisiae) cells areindependently combined with 10 μl of the various DNA mixtures from aboveand transferred to a 0.2-cm electroporation cuvette. The yeast/DNAmixtures are electropulsed using power supply (BioRad Laboratories,Hercules, Calif.) settings of 0.75 kV (5 kV/cm), ∞ ohms, 25 μF. To eachcuvette is added 600 μl of 1.2 M sorbitol, and the yeast is plated intwo 300-μl aliquots onto two URA-D plates and incubated at 30° C. Afterabout 48 hours, the Ura⁺ yeast transformants from a single plate areresuspended in 1 ml H₂O and spun briefly to pellet the yeast cells. Thecell pellet is resuspended in 1 ml of lysis buffer (2% Triton X-100, 1%SDS, 100 mM NaCl, 10 mM Tris, pH 8.0, 1 mM EDTA). Five hundredmicroliters of the lysis mixture is added to an Eppendorf tubecontaining 300 μl acid-washed glass beads and 200 μl phenol-chloroform,vortexed for 1 minute intervals two or three times, and spun for 5minutes in an Eppendorf centrifuge at maximum speed. Three hundredmicroliters of the aqueous phase is transferred to a fresh tube, and theDNA is precipitated with 600 μl ethanol (EtOH), followed bycentrifugation for 10 minutes at 4° C. The DNA pellet is resuspended in10 μl H₂O.

Transformation of electrocompetent E. coli host cells (Electromax DH10B™cells; obtained from Life Technologies, Inc., Gaithersburg, Md.) is donewith 0.5-2 ml yeast DNA prep and 40 μl of cells. The cells areelectropulsed at 1.7 kV, 25 μF, and 400 ohms. Following electroporation,1 ml SOC (2% Bacto™ Tryptone (Difco, Detroit, Mich.), 0.5% yeast extract(Difco), 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl₂, 10 mM MgSO₄, 20 mMglucose) is plated in 250-μl aliquots on four LB AMP plates (LB broth(Lennox), 1.8% Bacto™ Agar (Difco), 100 mg/L Ampicillin).

Individual clones harboring the correct expression construct for Zcyto21are identified by restriction digest to verify the presence of theZcyto21 insert and to confirm that the various DNA sequences have beenjoined correctly to one another. The inserts of positive clones aresubjected to sequence analysis. Larger scale plasmid DNA is isolatedusing a commercially available kit (QIAGEN Plasmid Maxi Kit, Qiagen,Valencia, Calif.) according to manufacturer's instructions. The correctconstruct is designated pZMP6/Zcyto21.

Example 2

CHO DG44 cells (Chasin et al., Som. Cell. Molec. Genet. 12:555-666,1986) are plated in 10-cm tissue culture dishes and allowed to grow toapproximately 50% to 70% confluency overnight at 37° C., 5% CO₂, inHam's F12/FBS media (Ham's F12 medium (Life Technologies), 5% fetalbovine serum (Hyclone, Logan, Utah), 1% L-glutamine (JRH Biosciences,Lenexa, Kans.), 1% sodium pyruvate (Life Technologies)). The cells arethen transfected with the plasmid Zcyto21/pZMP6 by liposome-mediatedtransfection using a 3:1 (w/w) liposome formulation of the polycationiclipid2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propaniminium-trifluoroacetateand the neutral lipid dioleoyl phosphatidylethanolamine inmembrane-filtered water (Lipofectamine™ Reagent, Life Technologies), inserum free (SF) media formulation (Ham's F12, 10 mg/ml transferrin, 5mg/ml insulin, 2 mg/ml fetuin, 1% L-glutamine and 1% sodium pyruvate).Zcyto21/pZMP6 is diluted into 15-ml tubes to a total final volume of 640μl with SF media. 35 μl of Lipofectamine™ is mixed with 605 μl of SFmedium. The resulting mixture is added to the DNA mixture and allowed toincubate approximately 30 minutes at room temperature. Five ml of SFmedia is added to the DNA:Lipofectamine™ mixture. The cells are rinsedonce with 5 ml of SF media, aspirated, and the DNA:Lipofectamine™mixture is added. The cells are incubated at 37° C. for five hours, then6.4 ml of Ham's F12/10% FBS, 1% PSN media is added to each plate. Theplates are incubated at 37° C. overnight, and the DNA:Lipofectamine™mixture is replaced with fresh 5% FBS/Ham's media the next day. On day 3post-transfection, the cells are split into T-175 flasks in growthmedium. On day 7 postransfection, the cells are stained withFITC-anti-CD8 monoclonal antibody (Pharmingen, San Diego, Calif.)followed by anti-FITC-conjugated magnetic beads (Miltenyi Biotec). TheCD8-positive cells are separated using commercially available columns(mini-MACS columns; Miltenyi Biotec) according to the manufacturer'sdirections and put into DMEM/Ham's F12/5% FBS without nucleosides butwith 50 nM methotrexate (selection medium).

Cells are plated for subcloning at a density of 0.5, 1 and 5 cells perwell in 96-well dishes in selection medium and allowed to grow out forapproximately two weeks. The wells are checked for evaporation of mediumand brought back to 200 μl per well as necessary during this process.When a large percentage of the colonies in the plate are nearconfluency, 100 μl of medium is collected from each well for analysis bydot blot, and the cells are fed with fresh selection medium. Thesupernatant is applied to a nitrocellulose filter in a dot blotapparatus, and the filter is treated at 100° C. in a vacuum oven todenature the protein. The filter is incubated in 625 mM Tris-glycine, pH9.1, 5 mM β-mercaptoethanol, at 65° C., 10 minutes, then in 2.5% non-fatdry milk Western A Buffer (0.25% gelatin, 50 mM Tris-HCl pH 7.4, 150 mMNaCl, 5 mM EDTA, 0.05% Igepal CA-630) overnight at 4° C. on a rotatingshaker. The filter is incubated with the antibody-HRP conjugate in 2.5%non-fat dry milk Western A buffer for 1 hour at room temperature on arotating shaker. The filter is then washed three times at roomtemperature in PBS plus 0.01% Tween 20, 15 minutes per wash. The filteris developed with chemiluminescence reagents (ECL™ direct labelling kit;Amersham Corp., Arlington Heights, Ill.) according to the manufacturer'sdirections and exposed to film (Hyperfilm ECL, Amersham Corp.) forapproximately 5 minutes. Positive clones are trypsinized from the96-well dish and transferred to 6-well dishes in selection medium forscaleup and analysis by Western blot.

Example 3

Full-length Zcyto21 protein is produced in BHK cells transfected withpZMP6/Zcyto21 (Example 1). BHK 570 cells (ATCC CRL-10314) are plated in10-cm tissue culture dishes and allowed to grow to approximately 50 to70% confluence overnight at 37° C., 5% CO₂, in DMEM/FBS media (DMEM,Gibco/BRL High Glucose; Life Technologies), 5% fetal bovine serum(Hyclone, Logan, Utah), 1 mM L-glutamine (JRH Biosciences, Lenexa,Kans.), 1 mM sodium pyruvate (Life Technologies). The cells are thentransfected with pZMP6/Zcyto21 by liposome-mediated transfection (usingLipofectamine™; Life Technologies), in serum free (SF) media (DMEMsupplemented with 10 mg/ml transferrin, 5 mg/ml insulin, 2 mg/ml fetuin,1% L-glutamine and 1% sodium pyruvate). The plasmid is diluted into15-ml tubes to a total final volume of 640 μl with SF media. 35 μl ofthe lipid mixture is mixed with 605 μl of SF medium, and the resultingmixture is allowed to incubate approximately 30 minutes at roomtemperature. Five milliliters of SF media is then added to theDNA:lipidmixture. The cells are rinsed once with 5 ml of SF media,aspirated, and the DNA:lipid mixture is added. The cells are incubatedat 37° C. for five hours, then 6.4 ml of DMEM/10% FBS, 1% PSN media isadded to each plate. The plates are incubated at 37° C. overnight, andthe DNA:lipid mixture is replaced with fresh 5% FBS/DMEM media the nextday. On day 5 post-transfection, the cells are split into T-162 flasksin selection medium (DMEM+5% FBS, 1% L-Gln, 1% NaPyr, 1 μMmethotrexate). Approximately 10 days post-transfection, two 150-mmculture dishes of methotrexate-resistant colonies from each transfectionare trypsinized, and the cells are pooled and plated into a T-162 flaskand transferred to large-scale culture.

Example 4

For construction of adenovirus vectors, the protein coding region ofhuman Zcyto21 is amplified by PCR using primers that add PmeI and AscIrestriction sites at the 5′ and 3′ termini respectively. Amplificationis performed with a full-length Zcyto21 cDNA template in a PCR reactionas follows: one cycle at 95° C. for 5 minutes; followed by 15 cycles at95° C. for 1 min., 61° C. for 1 min., and 72° C. for 1.5 min.; followedby 72° C. for 7 min.; followed by a 4° C. soak. The PCR reaction productis loaded onto a 1.2% low-melting-temperature agarose gel in TAE buffer(0.04 M Tris-acetate, 0.001 M EDTA). The Zcyto21 PCR product is excisedfrom the gel and purified using a commercially available kit comprisinga silica gel membrane spin column (QIAquick® PCR Purification Kit andgel cleanup kit; Qiagen, Inc.) as per kit instructions. The PCR productis then digested with PmeI and AscI, phenol/chloroform extracted, EtOHprecipitated, and rehydrated in 20 ml TE (Tris/EDTA pH 8). The Zcyto21fragment is then ligated into the PmeI-AscI sites of the transgenicvector pTG12-8 and transformed into E. coli DH10B™ competent cells byelectroporation. Vector pTG12-8 was derived from p2999B4 (Palmiter etal., Mol Cell Biol 13:5266-5275, 1993) by insertion of a rat insulin IIintron (ca. 200 bp) and polylinker (Fse I/Pme I/Asc I) into the Nru Isite. The vector comprises a mouse metallothionein (MT-1) promoter (ca.750 bp) and human growth hormone (hGH) untranslated region andpolyadenylation signal (ca. 650 bp) flanked by 10 kb of MT-1 5′ flankingsequence and 7 kb of MT-1 3′ flanking sequence. The cDNA is insertedbetween the insulin II and hGH sequences. Clones containing Zcyto21 areidentified by plasmid DNA miniprep followed by digestion with PmeI andAscI. A positive clone is sequenced to insure that there were nodeletions or other anomalies in the construct.

DNA is prepared using a commercially available kit (Maxi Kit, Qiagen,Inc.), and the Zcyto21 cDNA is released from the pTG12-8 vector usingPmeI and AscI enzymes. The cDNA is isolated on a 1% low meltingtemperature agarose gel and excised from the gel. The gel slice ismelted at 70 μC, and the DNA is extracted twice with an equal volume ofTris-buffered phenol, precipitated with EtOH, and resuspended in 10 μlH₂O.

The Zcyto21 cDNA is cloned into the EcoRV-AscI sites of a modifiedpAdTrack-CMV (He, T-C. et al., Proc. Natl. Acad. Sci. USA 95:2509-2514,1998). This construct contains the green fluorescent protein (GFP)marker gene. The CMV promoter driving GFP expression is replaced withthe SV40 promoter, and the SV40 polyadenylation signal is replaced withthe human growth hormone polyadenylation signal. In addition, the nativepolylinker is replaced with FseI, EcoRV, and AscI sites. This modifiedform of pAdTrack-CMV is named pZyTrack. Ligation is performed using acommercially available DNA ligation and screening kit (Fast-Link® kit;Epicentre Technologies, Madison, Wis.). Clones containing Zcyto21 areidentified by digestion of mini prep DNA with FseI and AscI. In order tolinearize the plasmid, approximately 5 μg of the resulting pZyTrackZcyto21 plasmid is digested with PmeI. Approximately 1 μg of thelinearized plasmid is cotransformed with 200 ng of supercoiled pAdEasy(He et al., ibid.) into E. coli BJ5183 cells (He et al., ibid.). Theco-transformation is done using a Bio-Rad Gene Pulser at 2.5 kV, 200ohms and 25 μFa. The entire co-transformation mixture is plated on 4 LBplates containing 25 μg/ml kanamycin. The smallest colonies are pickedand expanded in LB/kanamycin, and recombinant adenovirus DNA isidentified by standard DNA miniprep procedures. The recombinantadenovirus miniprep DNA is transformed into E. coli DH10B™ competentcells, and DNA is prepared using a Maxi Kit (Qiagen, Inc.) according tokit instructions.

Approximately 5 μg of recombinant adenoviral DNA is digested with PacIenzyme (New England Biolabs) for 3 hours at 37° C. in a reaction volumeof 100 μl containing 20-30U of PacI. The digested DNA is extracted twicewith an equal volume of phenol/chloroform and precipitated with ethanol.The DNA pellet is resuspended in 10 μl distilled water. A T25 flask ofQBI-293A cells (Quantum Biotechnologies, Inc. Montreal, Qc. Canada),inoculated the day before and grown to 60-70% confluence, is transfectedwith the PacI digested DNA. The PacI-digested DNA is diluted up to atotal volume of 50 μl with sterile HBS (150 mM NaCl, 20 mM HEPES). In aseparate tube, 20 μl of 1 mg/mlN-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium salts (DOTAP)(Boehringer Mannheim, Indianapolis, Ind.) is diluted to a total volumeof 100 μl with HBS. The DNA is added to the DOTAP, mixed gently bypipeting up and down, and left at room temperature for 15 minutes. Themedia is removed from the 293A cells and washed with 5 ml serum-freeminimum essential medium (MEM) alpha containing 1 mM sodium pyruvate,0.1 mM MEM non-essential amino acids, and 25 mM HEPES buffer (reagentsobtained from Life Technologies, Gaithersburg, Md.). 5 ml of serum-freeMEM is added to the 293A cells and held at 37° C. The DNA/lipid mixtureis added drop-wise to the T25 flask of 293A cells, mixed gently, andincubated at 37° C. for 4 hours. After 4 hours the media containing theDNA/lipid mixture is aspirated off and replaced with 5 ml complete MEMcontaining 5% fetal bovine serum. The transfected cells are monitoredfor GFP expression and formation of foci (viral plaques).

Seven days after transfection of 293A cells with the recombinantadenoviral DNA, the cells express the GFP protein and start to form foci(viral “plaques”). The crude viral lysate is collected using a cellscraper to collect all of the 293A cells. The lysate is transferred to a50-ml conical tube. To release most of the virus particles from thecells, three freeze/thaw cycles are done in a dry ice/ethanol bath and a37° C. waterbath.

The crude lysate is amplified (Primary (10) amplification) to obtain aworking “stock” of Zcyto21 rAdV lysate. Ten 10 cm plates of nearlyconfluent (80-90%) 293A cells are set up 20 hours previously, 200 ml ofcrude rAdV lysate is added to each 10-cm plate, and the cells aremonitored for 48 to 72 hours for CPE (cytopathic effect) under the whitelight microscope and expression of GFP under the fluorescent microscope.When all of the 293A cells show CPE, this stock lysate is collected andfreeze/thaw cycles performed as described above.

A secondary (2°) amplification of Zcyto21 rAdV is then performed. Twenty15-cm tissue culture dishes of 293A cells are prepared so that the cellsare 80-90% confluent. All but 20 ml of 5% MEM media is removed, and eachdish is inoculated with 300-500 ml of the 1° amplified rAdv lysate.After 48 hours the 293A cells are lysed from virus production, thelysate is collected into 250-ml polypropylene centrifuge bottles, andthe rAdV is purified.

NP-40 detergent is added to a final concentration of 0.5% to the bottlesof crude lysate in order to lyse all cells. Bottles are placed on arotating platform for 10 minutes agitating as fast as possible withoutthe bottles falling over. The debris is pelleted by centrifugation at20,000×G for 15 minutes. The supernatant is transferred to 250-mlpolycarbonate centrifuge bottles, and 0.5 volume of 20% PEG8000/2.5 MNaCl solution is added. The bottles are shaken overnight on ice. Thebottles are centrifuged at 20,000×G for 15 minutes, and the supernatantis discarded into a bleach solution. Using a sterile cell scraper, thewhite, virus/PEG precipitate from 2 bottles is resuspended in 2.5 mlPBS. The resulting virus solution is placed in 2-ml microcentrifugetubes and centrifuged at 14,000×G in the microcentrifuge for 10 minutesto remove any additional cell debris. The supernatant from the 2-mlmicrocentrifuge tubes is transferred into a 15-ml polypropylene snapcaptube and adjusted to a density of 1.34 g/ml with CsCl. The solution istransferred to 3.2-ml, polycarbonate, thick-walled centrifuge tubes andspun at 348,000 X G for 3-4 hours at 25 μC. The virus forms a whiteband. Using wide-bore pipette tips, the virus band is collected.

A commercially available ion-exchange columns (e.g., PD-10 columnsprepacked with Sephadex® G-25M; Pharmacia Biotech, Piscataway, N.J.) isused to desalt the virus preparation. The column is equilibrated with 20ml of PBS. The virus is loaded and allowed to run into the column. 5 mlof PBS is added to the column, and fractions of 8-10 drops arecollected. The optical densities of 1:50 dilutions of each fraction aredetermined at 260 nm on a spectrophotometer. Peak fractions are pooled,and the optical density (OD) of a 1:25 dilution is determined. OD isconverted to virus concentration using the formula: (OD at 260nm)(25)(1.1×10¹²)=virions/ml.

To store the virus, glycerol is added to the purified virus to a finalconcentration of 15%, mixed gently but effectively, and stored inaliquots at −80 μC.

A protocol developed by Quantum Biotechnologies, Inc. (Montreal, Canada)is followed to measure recombinant virus infectivity. Briefly, two96-well tissue culture plates are seeded with 1×10⁴ 293A cells per wellin MEM containing 2% fetal bovine serum for each recombinant virus to beassayed. After 24 hours 10-fold dilutions of each virus from 1×10⁻² to1×10⁻¹⁴ are made in MEM containing 2% fetal bovine serum. 100 μl of eachdilution is placed in each of 20 wells. After 5 days at 37° C., wellsare read either positive or negative for CPE, and a value for “PlaqueForming Units/ml” (PFU) is calculated.

Example 5

Trangenic animals expressing Zcyto21 genes are producing using adult,fertile males (studs) (B6C3f1, 2-8 months of age (Taconic Farms,Germantown, N.Y.)), vasectomized males (duds) (CD1, 2-8 months, (TaconicFarms)), prepubescent fertile females (donors) (B6C3f1, 4-5 weeks,(Taconic Farms)) and adult fertile females (recipients) (CD1, 2-4months, (Taconic Farms)).

The donors are acclimated for 1 week and then injected withapproximately 8 IU/mouse of Pregnant Mare's Serum gonadotrophin (Sigma,St. Louis, Mo.) I.P., and 46-47 hours later, 8 IU/mouse of humanChorionic Gonadotropin (hCG (Sigma)) I.P. to induce superovulation.Donors are mated with studs subsequent to hormone injections. Ovulationgenerally occurs within 13 hours of hCG injection. Copulation isconfirmed by the presence of a vaginal plug the morning followingmating.

Fertilized eggs are collected under a surgical scope (Leica MZ12 StereoMicroscope, Leica, Wetzlar, Del.). The oviducts are collected and eggsare released into urinanalysis slides containing hyaluronidase (Sigma).Eggs are washed once in hyaluronidase, and twice in Whitten's W640medium (Table 4) that has been incubated with 5% CO₂, 5% O₂, and 90% N₂at 37° C. The eggs are then stored in a 37° C./5% CO₂ incubator untilmicroinjection.

10-20 micrograms of plasmid DNA containing a cDNA of the Zcyto21gene islinearized, gel-purified, and resuspended in 10 mM Tris pH 7.4, 0.25 mMEDTA pH 8.0, at a final concentration of 5-10 nanograms per microliterfor microinjection.

Plasmid DNA is microinjected into harvested eggs contained in a drop ofW640 medium overlaid by warm, CO₂-equilibrated mineral oil. The DNA isdrawn into an injection needle (pulled from a 0.75 mm ID, 1 mm ODborosilicate glass capillary), and injected into individual eggs. Eachegg is penetrated with the injection needle, into one or both of thehaploid pronuclei.

Picoliters of DNA are injected into the pronuclei, and the injectionneedle withdrawn without coming into contact with the nucleoli. Theprocedure is repeated until all the eggs are injected. Successfullymicroinjected eggs are transferred into an organ tissue-culture dishwith pregassed W640 medium for storage overnight in a 37° C./5% CO₂incubator.

The following day, 12-17 healthy 2-cell embryos from the previous day'sinjection are transferred into the recipient. The swollen ampulla islocated and holding the oviduct between the ampulla and the bursa, anick in the oviduct is made with a 28 g needle close to the bursa,making sure not to tear the ampulla or the bursa. The embryos areimplanted through this nick, and by holding onto the peritoneal wall,the reproductive organs are guided back into the abdominal cavity.

The recipients are returned to cages in pairs, and allowed 19-21 daysgestation. After birth, 19-21 days postpartum is allowed before weaning.The weaklings are sexed and placed into separate sex cages, and a 0.5 cmbiopsy (used for genotyping) is snipped off the tail with cleanscissors.

Genomic DNA is prepared from the tail snips using a Qiagen Dneasy kitfollowing the manufacturer's instructions. Genomic DNA is analyzed byPCR using primers designed to the human growth hormone (hGH) 3′ UTRportion of the transgenic vector. A region unique to the human sequencewas identified from an alignment of the human and mouse growth hormone3′ UTR DNA sequences, ensuring that the PCR reaction does not amplifythe mouse sequence. Primers which amplify a 368 base pair fragment ofhGH and primers which hybridize to vector sequences and amplify the cDNAinsert, are often used along with the hGH primers. In these experiments,DNA from animals positive for the transgene will generate two bands, a368 base pair band corresponding to the hGH 3′ UTR fragment and a bandof variable size corresponding to the cDNA insert.

Once animals are confirmed to be transgenic (TG), they may beback-crossed into an inbred strain by placing a TG female with awild-type male, or a TG male with one or two wild-type female(s). Aspups are born and weaned, the sexes are separated, and their tailssnipped for genotyping.

Analysis of the mRNA expression level of each transgene is done using anRNA solution hybridization assay or real-time PCR on an ABI Prism 7700(PE Applied Biosystems, Inc., Foster City, Calif.) followingmanufacturer's instructions. TABLE 5 WHITTEN'S 640 MEDIA mgs/200 mmgs/500/ml NaCl 1280 3200 KCl 72 180 KH₂PO₄ 32 80 MgSO₄•7H₂O 60 150Glucose 200 500 Ca²⁺ Lactate 106 265 K Penn 15 37.5 Streptomycin SO₄ 1025 NaHCO₃ 380 950 Na Pyruvate 5 12.5 H₂0 200 500 EDTA 100 μl 250 μl 5%Phenol Red 200 μl 500 μl BSA 600 1500All reagents are available from Sigma.

Example 6 1. Stimulation of Expression from an Interferon-ResponsivePromoter

In one series of experiments, conditioned medium (CM) containing Zcyto21protein is generated by infecting 293A cells with recombinant adenoviruscontaining the cDNA for Zcyto21 (AdZy-Zcyto21) at a multiplicity ofinfection of 400 particles per cell. CM is harvested at time pointsbetween 40 hours post infection and stored at −20° C. CM is alsogenerated from an infection with a recombinant adenovirus lacking a cDNA(AdZy-parental). Prior to use, a portion of the CM is concentrated 14fold in a Millipore Ultrafree-15 (5,000 nominal molecular weight limit)centrifugal filter, and then, filtered through a Millipore Ultrafree-15(100,000 nominal molecular weight limit) centrifugal filter to reducethe amount of viral particles present in the media, and finally filteredthrough a Millipore 0.2 μm syringe filter to sterilize the CM.Concentrated CM samples are diluted 1:2 in binding buffer and incubatedwith cells from a murine cell line for 5 hours at 37° C.

2. Anti-Viral Activity of Zcyto21

Another series of experiments examines the anti-viral activity ofZcyto21. In these studies, the anti-viral assay is performed by platingL929 cells (ATCC No. CCL-1) in growth media RPMI medium 1640 containing10% fetal bovine serum, penicillin, streptomycin, and L-glutamine in96-well format at 50,000 cells per well. Adenovirus CM from 293A cellsinfected with either AdZy-Zcyto21m or AdZy-parental, as described above,are incubated with cells overnight. A positive control in the assay isprovided by murine interferon-α serially diluted 1:10, starting at 100ng/ml. L929 cells with growth media alone provided the negative control.Treated cells are incubated for 24 hours. The media are discarded, freshmedium are added, and encephalomyocarditis virus (ATCC No. vr129b) isintroduced at a multiplicity of infection of 0.1 (i.e., one virusparticle for every ten L929 cells). The cells are incubated in thepresence of the virus for 24 hours, and then, the wells are scored forpercent cytopathic effect (CPE

3. Antiproliferation Assay Using a BAF3 Cell Line

BaF3 is used to determine if Zcyto21 has anti-proliferative properties.Baby hamster kidney (BHK) cells are stably transfected with anexpression vector containing the CMV promoter plus intron A upstream ofthe Zcyto21 cDNA or an unrelated cDNA, called Zα30, using BRLlipofectamine. Stably transfected cells are seeded in a cell factory inserum free media and allowed to grow for three days before conditionedmedia is harvested and concentrated in a 5K filter to 10×. Concentratedconditioned medium samples are stored at 4° C.

The following assay is used to test for anti-proliferation of BaF3. In a96 well plate, eight 1:2 serial dilutions are made of growth media alone(RPMI 1640, 10% fetal bovine serum, 1 mM sodium pyruvate, 2 mML-glutamine), or murine IL-3 (starting at 50 pg/ml in growth medium)with final volume of 100 μl. Fifty microliters of the following areadded to both growth media alone or mIL-3 diluted lanes: humaninterferon-α (100 ng/ml, 10 ng/ml, or 1 ng/ml diluted in growth medium),human interferon-β (100 ng/ml, 10 ng/ml, or 1 ng/ml diluted in growthmedium), murine interferon-α (100 ng/ml, 10 ng/ml, or 1 ng/ml diluted ingrowth medium), murine interferon-β (100 ng/ml, 10 ng/ml, or 1 ng/mldiluted in growth medium), Zcyto21 (at 2.5×, 0.5×, or 0.1×), and murineZα30 (at 2.5×, 0.5×, or 0.1×).

The BaF3cell line is washed three times in growth medium, pellets areresuspended in growth medium, cells are counted and diluted in growthmedium to 5,000 cells/50 μl. Fifty microliters of diluted cells are thenadded to each dilution of samples. Assay plates are incubated in a 37°C. incubator for three to four days. Twenty microliters of Alomar blueare then added to each well and the plate are incubated overnight at 37°C. The plates are read on the fluorescent plate reader at excitationwavelength of 544 and emission wavelength 590.

Example 7 Tissue Distribution of Human Zcyto21 in Tissue Panels UsingPCR

A panel of cDNA samples from human tissues was screened for Zcyto21expression using PCR. The panel was made in house and contained 77marathon cDNA and cDNA samples from various normal and cancerous humantissues and cell lines as shown in Table 6, below. The cDNA samples camefrom in-house libraries or marathon cDNA preparations of RNA that wereprepared in-house, or from a commercial supplier such as Clontech (PaloAlto, Calif.) or Invitrogen (Carlsbad, Calif.). The marathon cDNAs weremade using the Marathon cDNA Amplification Kit (Clontech). To assurequality of the panel samples, three tests for quality control (QC) wererun: (1) To assess the RNA quality used for the libraries, the in-housecDNAs were tested for average insert size by PCR with vector oligos thatwere specific for the vector sequences for an individual cDNA library;(2) Standardization of the concentration of the cDNA in panel sampleswas achieved using standard PCR methods to amplify full length alphatubulin or G3PDH cDNA; and (3) a sample was sent to sequencing to checkfor possible ribosomal or mitochondrial DNA contamination. The panel wasset up in a 96-well format that included a human genomic DNA (Clontech)positive control sample. Each well contained approximately 0.2-100 pg/μlof cDNA. The first PCR reactions were set up using oligos ZC39,270 (SEQID NO:14) and ZC39,272 (SEQ ID NO:15), Advantage 2 DNA Polymerase Mix(Clontech) and Rediload dye (Research Genetics, Inc., Huntsville, Ala.).The amplification was carried out as follows: 1 cycle at 94° for 1minute then 35 cycles of 94° 10 seconds; 67°, 45 seconds and ended witha 3 minute final extension at 72°. The correct DNA fragment size wasobserved in brain, islet, prostate, testis, pituitary, placenta, ovariantumor, lung tumor, CD3+ and HPVS. Another PCR reaction was set up usingoligos ZC39,270 (SEQ ID NO:14) and ZC39,271 (SEQ ID NO:16), Advangtage 2DNA Polymerase Mix (Clontech) and Rediload dye (Research Genetics). Theamplification was carried out as follows: 1 cycle at 94°, 1 minute then35 cycles of 94°, 10 seconds; 65°, 30 seconds, 72°, 30 seconds and endedwith a 3 minute extension at 72°. The correct DNA fragment size wasobserved in pituitary, rectal tumor and ovarian tumor. TABLE 6 #samples#samples Tissue tested Tissue tested adrenal gland 1 bladder 1 bonemarrow 3 brain 2 cervix 1 colon 1 fetal brain 3 fetal heart 2 fetalkidney 1 fetal liver 2 fetal lung 1 fetal skin 1 heart 2 fetal muscle 1kidney 2 liver 1 lung 1 lymph node 1 mammary gland 1 melanoma 1 ovary 1pancreas 1 pituitary 2 placenta 3 prostate 3 rectum 1 salivary gland 2skeletal muscle 1 small intestine 1 spinal cord 2 spleen 1 uterus 1stomach 1 adipocyte library 1 testis 5 islet 1 thymus 1 prostate SMC 1thyroid 2 RPMI 1788 1 (ATCC # CCL-156) trachea 1 WI38 1 (ATCC # CCL-75)esophageal tumor 1 lung tumor 1 liver tumor 1 ovarian tumor 1 rectaltumor 1 stomach tumor 1 uterine tumor 2 CD3+ library 1 selected PBMC's(stimulated) HaCAT library 1 HPV library 1 (ATCC # CRL-2221) HPVSlibrary 1 MG63 library 1 (ATCC # CRL-2221) - selected K562 1(ATCC#CCL-243)

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

1. An isolated nucleic acid molecule encoding a zcyto21 polypeptidewherein the encoded polypeptide comprises amino acid residues 20-200 ofSEQ ID NO:5.
 2. The isolated nucleic acid molecule of claim 1 whereinthe encoded zcyto21 polypeptide comprises amino acid residues 1-200 ofSEQ ID NO:5.
 3. An expression vector comprising the isolated nucleicacid molecule of claim 1, a transcription promoter, and a transcriptionterminator, wherein the promoter is operably linked with the nucleicacid molecule, and wherein the nucleic acid molecule is operably linkedwith the transcription terminator.
 4. A recombinant host cell comprisingthe expression vector of claim 3, wherein the host cell is selected fromthe group consisting of bacterium, yeast cell, fungal cell, insect cell,mammalian cell, and plant cell.
 5. A method of producing a zcyto21protein, the method comprising the step of culturing recombinant hostcells of claim 4 under conditions suitable for the expression of theprotein.
 6. The method of claim 5 further comprising the step ofisolating the zcyto21 protein from the cultured recombinant host cells.